Human oncogene induced secreted protein I

ABSTRACT

The present invention relates to a novel protein, the Human Oncogene Induced Secreted Protein I (“HOIPS I”) protein. In particular, isolated nucleic acid molecules are provided encoding the human HOIPS I protein. HOIPS I polypeptides are also provided as are vectors, host cells and recombinant methods for producing the same. Also provided are diagnostic methods for detecting abnormal cell proliferation and differentiation disorders and therapeutic methods for treating the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application hereby claims priority benefit to U.S. appl.Ser. No. 60/033,869, filed Dec. 20, 1996 and U.S. appl. Ser. No.60/037,388, filed Feb. 7, 1997, which are hereby incorporated byreference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] Isolated nucleic acid molecules are provided encoding a humanoncogene induced secreted protein I (HOIPS I). HOIPS I polypeptides arealso provided, as are vectors, host cells and recombinant methods forproducing the same. Also provided are diagnostic methods for detectingmyeloid cells expressing the HOIPS I gene and therapeutic methods fortreating cell-proliferative diseases.

[0004] 2. Related Art

[0005] Hematopoiesis is the development and formation of blood cells inthe bone marrow, and is critical to the proper functioning of the immuneresponse. Differentiation of the myeloid cell lineage (granulocytes andmonocytes/macrophages) termed myelopoiesis commences in the human fetusat approximately six weeks of gestation. In the early stages ofmyelopoiesis, colony-forming units for granulocytes/monocytes (CFU-GMs)can be induced along either the granulocyte or monocyte pathways.Induction of the CFU-GM's along the granulocyte pathway results indistinct morphological stages of development, ultimately terminating inthe characteristic trilobed structure of polymorphonuclear leukocytes,also known as granulocytes.

[0006] Induction of CFU-GMs along the monocyte pathway gives riseinitially to proliferating monoblasts. Monoblasts differentiate intopromonocytes and, ultimately, into mature monocytes. Monocytes areconsidered to be circulating immature macrophages, which are highlydifferentiated cells found in various tissues.

[0007] Monocyte-macrophages are known to secrete a number ofbiologically active polypeptides called cytokines that affect thefunctions of other cells. Interleukin-1 (I-1), interleukin-6 (IL-6), andtumor necrosis factor-alpha (TNF-α) are all cytokines secreted bymonocytes/macrophages that play an important role in hematopoiesis.

[0008] A continued need exists for the further identification andcharacterization of the other cytokines and growth factors involved inhematopoiesis and immunoregulation.

[0009] Abnormal expression of the genes encoding the various cytokinesand growth factors involved in cell differentiation and proliferationcan result in neoplasias, including leukemias. Leukemia is defined as aprogressive malignant disease of the blood-forming organs, characterizedby distorted proliferation and development of leukocytes and theirprecursors in the blood and bone marrow. The leukemias account forapproximately 3 percent of all cancers in the United States. (Li, F. P.,“The Chronic Leukemias: Etiology and Epideriology,” in NeoplasticDiseases of the Blood, vol. I, pp. 7-17, Wiernik et al eds. (1985)).

[0010] Oncogenes have been implicated as a cause of human leukemias.Gelmann, E. P. et al., “The Etiology of Acute Leukemia: MolecularGenetics and Viral Oncology,” in Neoplastic Diseases of the Blood, vol.I, pp. 161-182, Wiernik et al. eds. (1985). An oncogene is a gene thatbrings about or contributes to neoplastic transformation of cells byencoding proteins which regulate cell growth and differentiation.Retroviral and cellular oncogenes arise from cellular genes calledproto-oncogenes, which appear to play an important role in normalhematopoietic cell growth and differentiation.

[0011] The isolation and characterization of viral oncogenes (v-onc)have facilitated the cloning and identification of the cellularoncogenes (c-onc) which derive their names from the respective viralgenes. They are highly conserved among species, and homologs are foundin all vertebrates, in lower organisms, and in humans. (Gelmann et al.)The role of c-onc genes in neoplasia has been investigated extensively.

[0012] The retroviral oncogene v-myb transforms myelomonocytichematopoietic cells in vivo and in vitro. (Moscovici, C. et al., Adv.Viral Oncol. 1:83-106 (1982)). The v-myb oncogene was originally definedby two naturally occurring avian retroviruses, AMV and E26, that inducemyeloid leukemias in chickens. (Moscovici et al.) The v-myb oncogenesare derived from a normal, cellular proto-oncogene, c-myb, which isexpressed in high levels in all immature hematopoietic lineages.(Kiempnauer, K. H. et al., Cell 31:537-547 (1984)). In contrast, v-myhoncogenes only transform a few cell types, such as the immature myeloidprecursors of neutrophils and macrophages. Both c-myb and v-myb encodenuclear, DNA binding proteins (i.e. transcription activators) thatregulate the phenotypes of normal and transformed hematopoietic cellsrespectively. (Ness, S. A. et al., Cell 59: 1115-1125 (1989); Burk, O.and Klempnauer, K. H., EMBO J. 10(12):3713-3719 (1991)). Thetransforming activity of these proteins is regulated by celltype-specific cofactors. The DNA-binding domain of the v-myb proteinscorresponds to the domain of several other myb-related DNA-bindingproteins isolated from such diverse species as mammals, insects, andplants. (Queva et aL 1992)

[0013] An interesting feature of the v-myb oncogene is that it not onlyblocks differentiation, but it also dictates the differentiationphenotype of the myeloid cells that it transforms. (Ness, S. A. et al.,Cell 59:1115-1125 (1989)). Expression of v-myb in myeloid cells resultsin them acquiring an immature phenotype. (Burk and Klempnauer, 1991). Inaddition, it has been shown that minor changes in the structure of thev-Myb protein determine whether the transformed cells take on thephenotype of immature macrophages or immature granulocytes, (Golay, J.et al., Cell 55:1147-1158 (1988)). Moreover, temperature-sensitive v-mybtransformed cells induced to differentiate can be induced toretrodifferentiate. (Introna, M. et al., Cell 63:1287-1297 (1990)).Different forms of v-myb impose alternate phenotypes of differentiationon transformed myeloid cells by regulating unique sets ofdifferentiation specific genes. (Introna, M. et al., Cell 63:1287-1297(1990)).

[0014] Two genes, identified as mim-1 and MD-1, are known to beregulated by v-myb. (Ness et al., 1989; Burk and Klempnauer, 1991). Themim-1 gene is specifically expressed in normal, immature, granulocytesand encodes a 35 kD secretable protein that is stored in the granules ofthose cells. (Ness et al., 1989; Queva, C. et al., Development114:125-133 (1992)). Indeed, mim-1 encodes one of the most abundantproteins found in granulocytes, and the high level of expressionsuggests that it may be a structural component of the promyelocytegranule. (Ness et al. 1989). When promyelocytes undergo terminaldifferentiation to neutrophil granulocytes, a decrease in the level ofmim-1 protein is observed. (Queva et al.) Moreover, analysis of chickembryo development shows that mim-1 mRNA transcripts are found wheregranulopoiesis occurs. (Queva et al.) Thus, mim-1 is the first describedmarker for cells that are differentiating into the granulocytic lineage.(Queva et al.; Introna et al.).

[0015] The mim-1 gene is one of a number v-myb-regulated genes thatcontribute to the unique differentiation phenotype displayed by bothnormal and transformed myeloid cells. Those genes, which include MD-1,must by definition be regulated similarly to mim- I by the various formsof the v-myb protein. (Ness et al.) It is likely that a number ofdifferent structural changes to the myb protein will alter the phenotypeof myeloid cells transformed by the v-myb oncogene and affect itscapacity to regulate its target genes, including mim-1 and MD-1.(Introna et al.).

[0016] Thus, v-myb acts as a master gene in hematopoietic celldifferentiation by regulating the expression of a unique set of geneswithin the myelomonocytic lineage. (Introna et al.) Because these genesare expected to be important regulators of cell differentiation andproliferation, their identification is critical to understanding themolecular mechanisms of neoplasia, transformation, and growth control.Thus, a need exists in the art for the identification of other genesinvolved in hematopoietic cell differentiation.

SUMMARY OF THE INVENTION

[0017] The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding the HOIPS I polypeptide having theamino acid sequence shown in FIG. 1 (SEQ ID NO:2) or the amino acidsequence encoded by the cDNA clone deposited in a bacterial host withthe American Type Culture Collection (“ATCC”), 12301 Park Lawn Drive,Rockville, Md. 20852, on Dec. 16, 1996. (ATCC Deposit Number 97825).

[0018] The present invention also relates to recombinant vectors, whichinclude the isolated nucleic acid molecules of the present invention,and to host cells containing the recombinant vectors, as well as tomethods of making such vectors and host cells and for using them forproduction of HOIPS I polypeptides or peptides by recombinanttechniques.

[0019] The invention further provides an isolated HOIPS I polypeptidehaving an amino acid sequence encoded by a polynucleotide describedherein.

[0020] In another embodiment, the present invention provides a methodfor inhibiting abnormal cell proliferation or differentiation byadministering to the abnormally proliferating or differentiating cell, asynthetic DNA or RNA construct of the present invention, wherein saidsynthetic DNA or RNA construct represses the functional expression ofthe HOIPS I gene. In an especially preferred embodiment, said DNAconstruct is operably linked to an inducible promoter.

[0021] In another embodiment, the present invention provides a methodfor identifying individuals who are believed to be predisposed to cellproliferative or differentiation disorders comprising the step ofidentifying individuals who have only one active allele of the HOIPS Igene.

[0022] The present invention provides a diagnostic method useful duringdiagnosis of a cell proliferative or cell differentiation disorder.

[0023] An additional aspect of the invention is related to a method fortreating an individual in need of an increased level of HOIPS I activityin the body comprising administering to such an individual a compositioncomprising a therapeutically effective amount of an isolated HOIPS Ipolypeptide of the invention or an agonist thereof.

[0024] A still further aspect of the invention is related to a methodfor treating an individual in need of a decreased level ofHOIPS Iactivity in the body comprising, administering to such an individual acomposition comprising a therapeutically effective amount of an HOIPS Iantagonist.

BRIEF DESCRIPTION OF THE FIGURES

[0025]FIG. 1 shows the nucleotide (SEQ ID NO: 1) and deduced amino acid(SEQ ID NO:2) sequences of HOIPS I. The protein has a leader sequence ofabout 20 amino acid residues and a deduced molecular weight of about17.8 kDa. The predicted amino acid sequence of the mature HOIPS Iprotein is also shown in FIG. 1 (SEQ ID NO:2).

[0026]FIG. 2 shows the regions of similarity between the amino acidsequences of the HOIPS I protein and chicken MD-1 (SEQ ID NO:3). Theconsensus sequence is shown (SEQ ID NO: 17).

[0027]FIG. 3 shows an analysis of the HOIPS I amino acid sequence.Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity;amphipathic regions; flexible regions; antigenic index and surfaceprobability are shown. In the “Antigenic Index—Jameson-Wolf” graph,amino acid residues about 17 to about 29, about 33 to about 39, about 43to about 52, about 56 to about 67, about 74 to about 83, about 90 toabout 94, about 110 to about 120, about 125 to about 139, and about 145to about 152 in FIG. 1 correspond to the shown highly antigenic regionsof the HOIPS I protein. These highly antigenic fragments in FIG. 1correspond to the following fragments, respectively in SEQ ID NO:2:amino acid residues about 4 to about 9, about 13 to about 19, about 23to about 32, about 36 to about 47, about 54 to about 63, about 70 toabout 74, about 90 to about 100, about 105 to about 119, and about 125to about 132.

DETAILED DESCRIPTION

[0028] The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding a HOIPS I polypeptide having theamino acid sequence shown in FIG. 1 (SEQ ID NO:2), which was determinedby sequencing a cloned cDNA. The HOIPS I protein of the presentinvention shares sequence homology with the chicken MD-1 protein. (FIG.2) (SEQ ID NO:3). The nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1)was obtained by sequencing the HTOCD71× clone, which was deposited onDec. 16, 1996 at the American Type Culture Collection, 12301 Park LawnDrive, Rockville, Md. 20852. (ATCC accession number 97825) The depositedclone is contained in the pbluescript SK(−) plasmid (Stratagene,LaJolla, Calif.).

[0029] Nucleic Acid Molecules

[0030] Unless otherwise indicated, all nucleotide sequences determinedby sequencing a DNA molecule herein were determined using an automatedDNA sequencer (such as the Model 373 from Applied Biosystems, Inc.), andall amino acid sequences of polypeptides encoded by DNA moleculesdetermined herein were predicted by translation of a DNA sequencedetermined as above. Therefore, as is known in the art for any DNAsequence determined by this automated approach, any nucleotide sequencedetermined herein may contain some errors. Nucleotide sequencesdetermined by automation are typically at least about 90% identical,more typically at least about 95% to at least about 99.9% identical tothe actual nucleotide sequence of the sequenced DNA molecule. The actualsequence can be more precisely determined by other approaches includingmanual DNA sequencing methods well known in the art. As is also known inthe art, a single insertion or deletion in a determined nucleotidesequence compared to the actual sequence will cause a frame shift intranslation of the nucleotide sequence such that the predicted aminoacid sequence encoded by a determined nucleotide sequence will becompletely different from the amino acid sequence actually encoded bythe sequenced DNA molecule, beginning at the point of such an insertionor deletion.

[0031] Using the information provided herein, such as the nucleotidesequence in FIG. 1, a nucleic acid molecule of the present inventionencoding a HOIPS I polypeptide may be obtained using standard cloningand screening procedures, such as those for cloning cDNAs using MRNA asstarting material. Illustrative of the invention, the nucleic acidmolecule described in FIG. 1 (SEQ ID NO: 1) was discovered in a cDNAlibrary derived from human tonsils tissue. The gene was also identifiedin cDNA libraries from the following tissues: bone marrow, dendriticcells, fetal and adult brain macrophages, B cells, and lymph nodes. Thedetermined nucleotide sequence of the HOIPS I cDNA of FIG. 1 (SEQ ID)NO: 1) contains an open reading frame encoding a protein of 162 aminoacid residues and a deduced molecular weight of about 17.8 kDa. TheHOIPS I protein shown in FIG. 1 (SEQ ID NO:2) is about 45% identical to,and about 64% similar to, the chicken MD-I protein (FIG. 2) in a 132amino acid residue overlap.

[0032] The present invention also provides the mature form(s) of theHOIPS I protein of the present invention. According to the signalhypothesis, proteins secreted by mammalian cells have a signal orsecretory leader sequence which is cleaved from the mature protein onceexport of the growing protein chain across the rough endoplasmicreticulum has been initiated. Most mammalian cells and even insect cellscleave secreted proteins with the same specificity. However, in somecases, cleavage of a secreted protein is not entirely uniform, whichresults in two or more mature species on the protein. Further, it haslong been known that the cleavage specificity of a secreted protein isultimately determined by the primary structure of the complete protein,that is, it is inherent in the amino acid sequence of the polypeptide.Therefore, the present invention provides a nucleotide sequence encodingthe mature HOIPS I polypeptides having the amino acid sequence encodedby the cDNA clone contained in the host deposited with the ATCC on Dec.16, 1996, (ATCC Deposit No. 97825) and as shown in FIG. 1 (SEQ ID NO:2).By the mature HOIPS I protein having the amino acid sequence encoded bythe cDNA clone contained in the host deposited with the ATCC on Dec. 16,1996, (ATCC Deposit No. 97825) is meant the mature form(s) of the HOIPSI protein produced by expression in a mammalian cell (e.g., COS cells,as described below) of the complete open reading frame encoded by thehuman DNA sequence of the clone contained in the vector in the depositedhost. As indicated below, the mature HOIPS I having the amino acidsequence encoded by the cDNA clone contained in the host deposited withthe ATCC on Dec. 16, 1996, (ATCC Deposit No. 97825) may or may notdiffer from the predicted “mature” HOIPS I protein shown in FIG. 1(amino acids from about 1 to about 142 in SEQ ID NO:2) depending on theaccuracy of the predicted cleavage site based on computer analysis.

[0033] Methods for predicting whether a protein has a secretory leaderas well as the cleavage point for that leader sequence are available.For instance, the methods of McGeoch (Virus Res. 3:271-286 (1985)) andvon Heinje (Nucleic Acids Res. 14:4683-4690 (1986)) can be used. Theaccuracy of predicting the cleavage points of known mammalian secretoryproteins for each of these methods is in the range of 75-80%. vonHeinje, supra. However, the two methods do not always produce the samepredicted cleavage point(s) for a given protein.

[0034] In the present case, the predicted amino acid sequence of thecomplete HOIPS I polypeptides of the present invention were analyzed bya computer program (“PSORT”) (K. Nakai and M. Kanehisa, Genomics14:897-911 (1992)), which is an expert system for predicting thecellular location of a protein based on the amino acid sequence. As partof this computational prediction of localization, the methods of McGeochand von Heinje are incorporated. The analysis by the PSORT programpredicted the cleavage sites between amino acids 20 and 21 in FIG. 1(SEQ ID NO:2). Thereafter, the complete amino acid sequences werefurther analyzed by visual inspection, applying a simple form of the(−1,−3) rule of von Heinje. von Heinje, supra. Thus, the leader sequencefor the HOIPS I protein is predicted to consist of amino acid residues−20 to −1 in SEQ ID NO:2. However, while the predicted mature HOIPS Iprotein consists of residues 1-142, the present inventors haveidentified other possible cleavage sites resulting in mature proteinshaving the following amino acid residues shown in SEQ ID NO:2: −7-142,−6-142, −5-142, −4-142, −3-142, −2-142, −1-142, 2-142, 3-142, 4-142,5-142, 6-142, 7-142, 8-142, 9-142, 10-142, 11-142, 12-142, 13-142,14-142.

[0035] As one of ordinary skill would appreciate, due to thepossibilities of sequencing errors discussed above, as well as thevariability of cleavage sites for leaders in different known proteins,the predicted HOIPS I polypeptide encoded by the deposited cDNAcomprises about 162 amino acids, but may be anywhere in the range of142-182 amino acids; and the predicted leader sequence of this proteinis about 20 amino acids, but may be anywhere in the range of about 13 toabout 33 amino acids.

[0036] As indicated, nucleic acid molecules of the present invention maybe in the form of RNA, such as mRNA, or in the form of DNA, including,for instance, cDNA and genomic DNA obtained by cloning or producedsynthetically. The DNA may be double-stranded or single-stranded.Single-stranded DNA or RNA may be the coding strand, also known as thesense strand, or it may be the non-coding strand, also referred to asthe anti-sense strand.

[0037] By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its native environmentFor example, recombinant DNA molecules contained in a vector areconsidered isolated for the purposes of the present invention. Furtherexamples of isolated DNA molecules include recombinant DNA moleculesmaintained in heterologous host cells or purified (partially orsubstantially) DNA molecules in solution. Isolated RNA molecules includein vivo or in vitro RNA transcripts of the DNA molecules of the presentinvention. Isolated nucleic acid molecules according to the presentinvention further include such molecules produced synthetically.

[0038] Isolated nucleic acid molecules of the present invention includeDNA molecules comprising an open reading frame (ORF) shown in FIG. 1(SEQ ID NO: 1); DNA molecules comprising the coding sequence for themature HOIEPS I protein shown in FIG. 1 (last 142 amino acids) (SEQ IDNO:2); and DNA molecules which comprise a sequence substantiallydifferent from those described above but which, due to the degeneracy ofthe genetic code, still encode the HOIPS I protein. Of course, thegenetic code is well known in the art. Thus, it would be routine for oneskilled in the art to generate such degenerate variants.

[0039] In another aspect, the invention provides isolated nucleic acidmolecules encoding the HOIPS I polypeptide having an amino acid sequenceas encoded by the cDNA clone contained in the plasmid deposited with theATCC on Dec. 16, 1996 (ATCC Deposit No. 97825). In a further embodiment,nucleic acid molecules are provided encoding the mature HOIPS Ipolypeptide or the full-length polypeptide lacking the N-terminalmethionine. The invention also provides an isolated nucleic acidmolecule having the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1)or the nucleotide sequence of the HOIPS I cDNA contained in theabove-described deposited clone, or a nucleic acid molecule having asequence complementary to one of the above sequences. Such isolatedmolecules, particularly DNA molecules, are useful as probes for genemapping, by in situ hybridization with chromosomes, and for detectingexpression of the HOIPS I gene in human tissue, for instance, byNorthern blot analysis.

[0040] The present invention is further directed to fragments of theisolated nucleic acid molecules described herein. By a fragment of anisolated nucleic acid molecule having the nucleotide sequence of thedeposited cDNA or the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1)is intended fragments at least about 15 nt, and more preferably at leastabout 20 nt, still more preferably at least about 30 nt, and even morepreferably, at least about 40 nt in length which are useful asdiagnostic probes and primers as discussed herein. Of course, largerfragments 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 nt inlength are also useful according to the present invention as arefragments corresponding to most, if not all, of the nucleotide sequenceof the deposited cDNA or as shown in FIG. 1 (SEQ ID NO: 1). By afragment at least 20 nt in length, for example, is intended fragmentswhich include 20 or more contiguous bases from the nucleotide sequenceof the deposited cDNA or the nucleotide sequence as shown in FIG. 1 (SEQID NO: 1).

[0041] Preferred nucleic acid fragments of the present invention includenucleic acid molecules encoding epitope-bearing portions of the HOIPS Iprotein. In particular, such nucleic acid fragments of the presentinvention include nucleic acid molecules encoding: a polypeptidecomprising amino acid residues from about −4 to about 9 of SEQ ID NO:2,a polypeptide comprising amino acid residues from about 13 to about 19of SEQ ID NO:2, a polypeptide comprising amino acid residues from about23 to about 32 of SEQ ID NO:2, a polypeptide comprising amino acidresidues from about 36 to about 47 of SEQ ID NO:2, a polypeptidecomprising amino acid residues from about 54 to about 63 of SEQ ID NO:2,a polypepfide comprising amino acid residues from about 70 to about 74of SEQ ID NO:2, a polypeptide comprising amino acid residues from about90 to about 100 of SEQ ID NO:2, a polypeptide comprising amino acidresidues from about 105 to about 119 of SEQ ID NO:2, and a polypeptidecomprising amino acid residues from about 125 to about 132 of SEQ IDNO:2. The inventors have determined that the above polypeptide fragmentsare antigenic regions of the HOIPS I protein. Methods for determiningother such epitope-bearing portions of the HOIPS I protein are describedin detail below.

[0042] In addition, the present inventors have identified the followingcDNA clone related to extensive portions of SEQ ID NO: 1: HCASG14R (SEQID NO: 11).

[0043] The following public ESTs, which relate to portions of SEQ ID NO:1, have also been identified: GenBank Accession No. AA340310 (SEQ ID NO:12); GenBank Accession No. T91708 (SEQ ID NO:13); GenBank Accession No.T92475 (SEQ ID NO: 14); GenBank Accession No. T84854 (SEQ ID NO: 15);and GenBank Accession No. C02431 (SEQ ID NO: 16).

[0044] In another aspect, the invention provides an isolated nucleicacid molecule comprising a polynucleotide which hybridizes understringent hybridization conditions to a portion of the polynucleotide ina nucleic acid molecule of the invention described above, for instance,the cDNA clone deposited with the ATCC on December 16, 1996 (ATCCDeposit No. 97825). By “stringent hybridization conditions” is intendedovernight incubation at 42° C. in a solution comprising: 50% formamide,5× SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate(pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 g/mldenatured, sheared salmon sperm DNA, followed by washing the filters in0.1× SSC at about 65° C.

[0045] By a polynucleotide which hybridizes to a “portion” of apolynucleotide is intended a polynucleotide (either DNA or RNA)hybridizing to at least about 15 nucleotides (nt), and more preferablyat least about 20 nt, still more preferably at least about 30 nt, andeven more preferably about 30-70 nt of the reference polynucleotide.These are useful as diagnostic probes and primers as discussed above andin more detail below.

[0046] By a portion of a polynucleotide of “at least 20 nt in length,”for example, is intended 20 or more contiguous nucleotides from thenucleotide sequence of the reference polynucleotide (e.g., the depositedcDNA or the nucleotide sequence as shown in FIG. 1 (SEQ ID NO: 1)). Ofcourse, a polynucleotide which hybridizes only to a poly A sequence(such as the 3′ terminal poly(A) tract of the HOIPS I cDNA shown in FIG.1 (SEQ ID NO: 1)), or to a complementary stretch of T (or U) residues,would not be included in a polynucleotide of the invention used tohybridize to a portion of a nucleic acid of the invention, since such apolynucleotide would hybridize to any nucleic acid molecule containing apoly (A) stretch or the complement thereof (e.g., practically anydouble-stranded cDNA clone).

[0047] As indicated, nucleic acid molecules of the present inventionwhich encode a HOIPS I polypeptide may include, but are not limited tothose encoding the amino acid sequence of the mature polypeptide, byitself; the coding sequence for the mature polypeptide and additionalsequences, such as those encoding the about 20 amino acid leader orsecretory sequence, such as a pre-, or pro- or prepro- protein sequence;the coding sequence of the mature polypeptide, with or without theaforementioned additional coding sequences, together with additional,non-coding sequences, including for example, but not limited to intronsand non-coding 5′ and 3′ sequences, such as the transcribed,non-translated sequences that play a role in transcription, mRNAprocessing, including splicing and polyadenylation signals, forexample—ribosome binding and stability of mRNA; an additional codingsequence which codes for additional amino acids, such as those whichprovide additional functionalities. Thus, the sequence encoding thepolypeptide may be fused to a marker sequence, such as a sequenceencoding a peptide which facilitates purification of the fusedpolypeptide. In certain preferred embodiments of this aspect of theinvention, the marker amino acid sequence is a hexa-histidine peptide,such as the tag provided in a pQE vector (Qiagen, Inc.), among others,many of which are commercially available. As described in Gentz et al.,Proc. Natl. Acad Sci. USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. The “HA” tag is another peptide useful for purification whichcorresponds to an epitope derived from the influenza hemagglutininprotein, which has been described by Wilson et al., Cell 37: 767 (1984).As discussed below, other such fusion proteins include the HOIPS I fusedto Fc at the N- or C-terminus.

[0048] The present invention further relates to variants of the nucleicacid molecules of the present invention, which encode portions, analogsor derivatives of the HOIPS I protein. Variants may occur naturally,such as a natural allelic variant. By an “allelic variant” is intendedone of several alternate forms of a gene occupying a given locus on achromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons,New York (1985). Non-naturally occurring variants may be produced usingart-known mutagenesis techniques.

[0049] Such variants include those produced by nucleotide substitutions,deletions or additions, which may involve one or more nucleotides. Thevariants may be altered in coding regions, non-coding regions, or both.Alterations in the coding regions may produce conservative ornon-conservative amino acid substitutions, deletions or additions.Especially preferred among these are silent substitutions, additions anddeletions, which do not alter the properties and activities of the HOIPSI protein or portions thereof Also especially preferred in this regardare conservative substitutions.

[0050] Further embodiments of the invention include isolated nucleicacid molecules comprising a polynucleotide having a nucleotide sequenceat least 95% identical, and more preferably at least 96%, 97%, 98% or99% identical to (a) a nucleotide sequence encoding the polypeptidehaving the amino acid sequence in SEQ ID NO:2; (b) a nucleotide sequenceencoding the polypeptide having the amino acid sequence in SEQ ID NO: 2,but lacking the N-terminal methionine; (c) a nucleotide sequenceencoding the polypeptide having the amino acid sequence at positionsfrom about 1 to about 142 in FIG. 1 SEQ ID NO:2; (d) a nucleotidesequence encoding the polypeptide having the amino acid sequence encodedby the cDNA clone contained in ATCC Deposit No. 97825; (e) a nucleotidesequence encoding the mature HOIPS I polypeptide having the amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No. 97825;or (f) a nucleotide sequence complementary to any of the nucleotidesequences in (a), (b), (c), (d), or (e).

[0051] By a polynucleotide having a nucleotide sequence at least, forexample, 95% “identical” to a reference nucleotide sequence encoding aHOIPS I polypeptide is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding the HOIPSI polypeptide. In other words, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. These mutations of thereference sequence may occur at the 5′ or 3′ terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence.

[0052] As a practical matter, whether any particular nucleic acidmolecule is at least 95%, 96%, 97%, 98% or 99% identical to, forinstance, the nucleotide sequence shown in SEQ ID NO: 1 or to thenucleotide sequence of the deposited cDNA clone can be determinedconventionally using known computer programs such as the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711. Bestfit uses the local homology algorithm of Smith andWaterman, Advances in Applied Mathematics 2: 482-489 (1981), to find thebest segment of homology between two sequences. When using Bestfit orany other sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set, of course,such that the percentage of identity is calculated over the full lengthof the reference nucleotide sequence and that gaps in homology of up to5% of the total number of nucleotides in the reference sequence areallowed.

[0053] The present application is directed to nucleic acid molecules atleast 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequenceshown in SEQ ID NO: 1 or to the nucleic acid sequence of the depositedcDNA, irrespective of whether they encode a polypeptide having HOIPS Iactivity. This is because even where a particular nucleic acid moleculedoes not encode a polypeptide having HOIPS I activity, one of skill inthe art would still know how to use the nucleic acid molecule, forinstance, as a hybridization probe or as a polymerase chain reaction(PCR) primer. Uses of the nucleic acid molecules of the presentinvention that do not encode a polypeptide having HOIPS I activityinclude, inter alia, (1) isolating the HOIPS I gene or allelic variantsthereof in a cDNA library; (2) in situ hybridization (e.g., “FISH”) tometaphase chromosomal spreads to provide precise chromosomal location ofthe HOIPS I gene, as described in Verma et al., Human Chromosomes: AManual of Basic Techniques, Pergamon Press, New York (1988); andNorthern Blot analysis for detecting HOIPS I mRNA expression in specifictissues.

[0054] Preferred, however, are nucleic acid molecules having sequencesat least 95%, 96%, 97%, 98% or 99% identical to the nucleic acidsequence shown in SEQ ID NO: 1 or to the nucleic acid sequence of thedeposited cDNA which do, in fact, encode a polypeptide having HOIPS Iprotein activity. By “a polypeptide having HOIPS I activity” is intendedpolypeptides exhibiting activity similar, but not necessarily identical,to an activity of the HOIPS I protein of the invention (either thefull-length protein or, preferably, the mature protein).

[0055] Of course, due to the degeneracy of the genetic code, one ofordinary skill in the art will immediately recognize that a large numberof the nucleic acid molecules having a sequence at least 95%, 96%, 97%,98%, or 99% identical to the nucleic acid sequence of the deposited cDNAor the nucleic acid sequence shown in SEQ ID NO: 1 will encode apolypeptide “having HOIPS I protein activity.” In fact, since degeneratevariants of these nucleotide sequences all encode the same polypeptide,this will be clear to the skilled artisan even without performing theabove described comparison assay. It will be further recognized in theart that, for such nucleic acid molecules that are not degeneratevariants, a reasonable number will also encode a polypeptide havingHOIPS I protein activity. This is because the skilled artisan is fullyaware of amino acid substitutions that are either less likely or notlikely to significantly effect protein function (e.g., replacing onealiphatic amino acid with a second aliphatic amino acid).

[0056] For example, guidance concerning how to make phenotypicallysilent amino acid substitutions is provided in Bowie, J. U. et al.,“Deciphering the Message in Protein Sequences: Tolerance to Amino AcidSubstitutions,” Science 247:1306-1310 (1990), wherein the authorsindicate that proteins are surprisingly tolerant of amino acidsubstitutions.

[0057] Vectors and Host Cells

[0058] The present invention also relates to vectors which include theisolated DNA molecules of the present invention, host cells which aregenetically engineered with the recombinant vectors, and the productionof HOIPS I polypeptides or fragments thereof by recombinant techniques.

[0059] The polynucleotides may be joined to a vector containing aselectable marker for propagation in a host. Generally, a plasmid vectoris introduced in a precipitate, such as a calcium phosphate precipitate,or in a complex with a charged lipid. If the vector is a virus, it maybe packaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

[0060] The DNA insert should be operatively linked to an appropriatepromoter, such as the phage lambda PL promoter, the E. coli lac, trp andtac promoters, the SV40 early and late promoters and promoters ofretroviral LTRs, to name a few. Other suitable promoters will be knownto the skilled artisan. The expression constructs will further containsites for transcription initiation, termination and, in the transcribedregion, a ribosome binding site for translation. The coding portion ofthe mature transcripts expressed by the constructs will preferablyinclude a translation initiating at the beginning and a terminationcodon (UAA, UGA or UAG) appropriately positioned at the end of thepolypeptide to be translated.

[0061] As indicated, the expression vectors will preferably include atleast one selectable marker. Such markers include dihydrofolatereductase or neomycin resistance for eukaryotic cell culture andtetracycline or ampicillin resistance genes for culturing in E. coli andother bacteria. Representative examples of appropriate hosts include,but are not limited to, bacterial cells, such as E. coli, Streptomycesand Salmonella typhimurium cells; fungal cells, such as yeast cells;insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animalcells such as CHO, COS and Bowes melanoma cells; and plant cells.Appropriate culture mediums and conditions for the above-described hostcells are known in the art.

[0062] Among vectors preferred for use in bacteria include pQE70, pQE60and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors,Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available fromStratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 availablefrom Pharmacia. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT,pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG andpSVL available from Pharmacia. Other suitable vectors will be readilyapparent to the skilled artisan.

[0063] Introduction of the construct into the host cell can be effectedby calcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986).

[0064] The polypeptide may be expressed in a modified form, such as afusion protein, and may include not only secretion signals, but alsoadditional heterologous functional regions. For instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N-terminus of the polypeptide to improve stability andpersistence in the host cell, during purification, or during subsequenthandling and storage. Also, peptide moieties may be added to thepolypeptide to facilitate purification. Such regions may be removedprior to final preparation of the polypeptide. The addition of peptidemoieties to polypeptides to engender secretion or excretion, to improvestability and to facilitate purification, among others, are familiar androutine techniques in the art. A preferred fusion protein comprises aheterologous region from immunoglobulin that is useful to solubilizeproteins. For example, EP-A-O 464 533 (Canadian counterpart 2045869)discloses fuision proteins comprising various portions of constantregion of immunoglobin molecules together with another human protein orpart thereof. In many cases, the Fc part in a fusion protein isthoroughly advantageous for use in therapy and diagnosis and thusresults, for example, in improved pharmacokinetic properties (EP-A 0232262). On the other hand, for some uses it would be desirable to be ableto delete the Fc part after the fusion protein has been expressed,detected and purified in the advantageous manner described. This is thecase when the Fc portion proves to be a hindrance to use in therapy anddiagnosis, for example when the fusion protein is to be used as antigenfor immunizations. In drug discovery, for example, human proteins, suchas hIL-5 have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. See,D. Bennett et al., Journal of Molecular Recognition, Vol. 852-58 (1995)and K. Johanson et al., The Journal of Biological Chemistry, Vol. 270,No. 16, pp 9459-9471 (1995).

[0065] The HOIPS I protein can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography (“BPLC”) is employed for purification.Polypeptides of the present invention include naturally purifiedproducts, products of chemical synthetic procedures, and productsproduced by recombinant techniques from a prokaryotic or eukaryotichost, including, for example, bacterial, yeast, higher plant, insect andmammalian cells. Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. In addition, polypeptides ofthe invention may also include an initial modified methionine residue,in some cases as a result of host-mediated processes.

[0066] HOIPS I Polypeptides and Fragments

[0067] The invention further provides an isolated HOIPS I polypeptidehaving the amino acid sequence encoded by the deposited cDNA, or theamino acid sequence in FIG. 1 (SEQ ID NO:2), or a peptide or polypeptidecomprising a portion of the above polypeptides.

[0068] It will be recognized in the art that some amino acid sequencesof the HOIPS I polypeptide can be varied without significant effect ofthe structure or function of the protein. If such differences insequence are contemplated, it should be remembered that there will becritical areas on the protein which determine activity.

[0069] Thus, the invention further includes variations of the HOIPS Ipolypeptide which show substantial HOIPS I polypeptide activity or whichinclude regions of HOIPS I protein such as the protein portionsdiscussed below. Such mutants include deletions, insertions, inversions,repeats, and type substitutions. As indicated above, guidance concerningwhich amino acid changes are likely to be phenotypically silent can befound in Bowie, J. U., et al., “Deciphering the Message in ProteinSequences: Tolerance to Amino Acid Substitutions,” Science 247:1306-1310(1990).

[0070] Thus, the fragment, derivative or analog of the polypeptide ofSEQ ID NO:2, or that encoded by the deposited cDNA, may be (i) one inwhich one or more of the amino acid residues are substituted with aconserved or non-conserved amino acid residue (preferably a conservedamino acid residue) and such substituted amino acid residue may or maynot be one encoded by the genetic code, or (ii) one in which one or moreof the amino acid residues includes a substituent group, or (iii) one inwhich the mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as an IgG Fc fusion regionpeptide or leader or secretory sequence or a sequence which is employedfor purification of the mature polypeptide or a proprotein sequence.Such fragments, derivatives and analogs are deemed to be within thescope of those skilled in the art from the teachings herein.

[0071] Of particular interest are substitutions of charged amino acidswith another charged amino acid and with neutral or negatively chargedamino acids. The latter results in proteins with reduced positive chargeto improve the characteristics of the HOIPS I protein. The prevention ofaggregation is highly desirable. Aggregation of proteins not onlyresults in a loss of activity but can also be problematic when preparingpharmaceutical formulations, because they can be immunogenic. (Pinckardet al., Clin Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes36:838-845 (1987); Cleland et al. Crit. Rev. Therapeutic Drug CarrierSystems 10:307-377 (1993)).

[0072] As indicated, changes are preferably of a minor nature, such asconservative amino acid substitutions that do not significantly affectthe folding or activity of the protein (see Table 1). TABLE 1Conservative Amino Acid Substitutions. Aromatic Phenylalanine TryptophanTyrosine Hydrophobic Leucine Isoleucine Valine Polar GlutamineAsparagine Basic Arginine Lysine Histidine Acidic Aspartic Acid GlutamicAcid Small Alanine Serine Threonine Methionine Glycine

[0073] Of course, the number of amino acid substitutions a skilledartisan would make depends on many factors, including those describedabove. Generally speaking, the number of amino acid substitutions forany given HOIPS I polypeptide will not be more than 50, 40, 30, 20, 10,5, or 3.

[0074] Amino acids in the HOIPS I protein of the present invention thatare essential for function can be identified by methods known in theart, such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244:1081-1085 (1989)). The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as in vitro proliferative activity.

[0075] The polypeptides of the present invention are preferably providedin an isolated form. By “isolated polypeptide” is intended a polypeptideremoved from its native environment. Thus, a polypeptide produced and/orcontained within a recombinant host cell is considered “isolated” forpurposes of the present invention. Also intended as an “isolatedpolypeptide” are polypeptides that have been purified, partially orsubstantially, from a recombinant host cell or from a native source. Forexample, a recombinantly produced version of the HOIPS I polypeptide canbe substantially purified by the one-step method described in Smith andJohnson, Gene 67:31-40 (1988).

[0076] The polypeptides of the present invention include the polypeptideencoded by the deposited cDNA including the leader, the maturepolypeptide encoded by the deposited cDNA minus the leader (i.e., themature protein), a polypeptide comprising amino acids about −20 to about142 in SEQ ID NO:2; a polypeptide comprising the amino acids about −19to about 142 in SEQ ID NO:2; a polypeptide comprising amino acids about1 to about 142 in SEQ ID NO:2; as well as polypeptides which are atleast 95% identical, more preferably at least 96%, 97%, 98% or 99%identical to those described above and also include portions of suchpolypeptides with at least 30 amino acids and more preferably at least50 amino acids.

[0077] By a polypeptide having an amino acid sequence at least, forexample, 95% “identical” to a reference amino acid sequence of a HOIPS Ipolypeptide is intended that the amino acid sequence of the polypeptideis identical to the reference sequence except that the polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the reference amino acid of the HOIPS I polypeptide. Inother words, to obtain a polypeptide having an amino acid sequence atleast 95% identical to a reference amino acid sequence, up to 5% of theamino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to 5%of the total amino acid residues in the reference sequence may beinserted into the reference sequence. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

[0078] As a practical matter, whether any particular polypeptide is atleast 95%, 96%, 97%, 98% or 99% identical to, for instance, the aminoacid sequence shown in SEQ ID NO:2 or to the amino acid sequence encodedby the deposited cDNA clone can be determined conventionally using knowncomputer programs such as the Bestfit program (Wisconsin SequenceAnalysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, 575 Science Drive, Madison, Wis. 53711. Whenusing Bestfit or any other sequence alignment program to determinewhether a particular sequence is, for instance, 95% identical to areference sequence according to the present invention, the parametersare set, of course, such that the percentage of identity is calculatedover the full length of the reference amino acid sequence and that gapsin homology of up to 5% of the total number of amino acid residues inthe reference sequence are allowed.

[0079] The polypeptides of the present invention are useful as amolecular weight marker on SDS-PAGE gels or on molecular sieve gelfiltration columns using methods well known to those of skill in theart.

[0080] In another aspect, the invention provides a peptide orpolypeptide comprising an epitope-bearing portion of a polypeptide ofthe invention. The epitope of this polypeptide portion is an immunogenicor antigenic epitope of a polypeptide described herein. An “immunogenicepitope” is defined as a part of a protein that elicits an antibodyresponse when the whole protein is the immunogen. On the other hand, aregion of a protein molecule to which an antibody can bind is defined asan “antigenic epitope.” The number of immunogenic epitopes of a proteingenerally is less than the number of antigenic epitopes. See, forinstance, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983).

[0081] As to the selection of peptides or polypeptides bearing anantigenic epitope (i.e., that contain a region of a protein molecule towhich an antibody can bind), it is well known in that art thatrelatively short synthetic peptides that mimic part of a proteinsequence are routinely capable of eliciting an antiserum that reactswith the partially mimicked protein. See, for instance, Sutcliffe, J.G., Shinnick, T. M., Green, N. and Learner, R. A. (1983) Antibodies thatreact with predetermined sites on proteins. Science 219:660-666.Peptides capable of eliciting protein-reactive sera are frequentlyrepresented in the primary sequence of a protein, can be characterizedby a set of simple chemical rules, and are confined neither toimmunodominant regions of intact proteins (i.e., immunogenic epitopes)nor to the amino or carboxyl terminals.

[0082] Antigenic epitope-bearing peptides and polypeptides of theinvention are therefore useful to raise antibodies, including monoclonalantibodies, that bind specifically to a polypeptide of the invention.See, for instance, Wilson et al, Cell 37:767-778 (1984) at 777.

[0083] Antigenic epitope-bearing peptides and polypeptides of theinvention preferably contain a sequence of at least seven, morepreferably at least nine and most preferably between at least about 15to about 30 amino acids contained within the amino acid sequence of apolypeptide of the invention.

[0084] Non-limiting examples of antigenic polypeptides or peptides thatcan be used to generate HOIPS I-specific antibodies include: apolypeptide comprising amino acid residues from about −4 to about 9 ofSEQ ID NO:2, a polypeptide comprising amino acid residues from about 13to about 19 of SEQ ID NO:2 , a polypeptide comp rising amino acidresidues from about 23 to about 32 of SEQ ID NO:2, a polypeptidecomprising amino acid residues from about 36 to about 47 of SEQ ID NO:2, a polypeptide comprising amino acid residues from about 54 to about 63of SEQ ID NO:2 , a polypeptide comprising amino acid residues from about70 to about 74 of SEQ ID NO:2, a polypeptide comprising amino acidresidues from about 90 to about 100 of SEQ ID NO:2, a polypeptidecomprising amino acid residues from about 105 to about 119 of SEQ IDNO:2, and a polypeptide comprising amino acid residues from about 125 toabout 132 of SEQ ID NO:2. As indicated above, the inventors havedetermined that the above polypeptide fragments are antigenic regions ofthe HOIPS I protein.

[0085] The epitope-bearing peptides and polypeptides of the inventionmay be produced by any conventional means. (Houghten, R. A., “Generalmethod for the rapid solid-phase synthesis of large numbers of peptides:specificity of antigen-antibody interaction at the level of individualamino acids,” Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985)). This“Simultaneous Multiple Peptide Synthesis (SMPS)” process is furtherdescribed in U.S. Pat. No. 4,631,211 to Houghten et al. (1986).

[0086] As one of skill in the art will appreciate, HOIPS I polypeptidesof the present invention and the epitope-bearing fragments thereofdescribed above can be combined with parts of the constant domain ofimmunoglobulins (IgG), resulting in chimeric polypeptides. These fusionproteins facilitate purification and show an increased half-life invivo. This has been shown, e.g., for chimeric proteins consisting of thefirst two domains of the human CD4-polypeptide and various domains ofthe constant regions of the heavy or light chains of mammalianimmunoglobulins (EPA 394,827; Traunecker et al., Nature 331:84-86(1988)). Fusion proteins that have a disulfide-linked dimeric structuredue to the IgG part can also be more efficient in binding andneutralizing other molecules than the monomeric HOIPS I protein orprotein fragment alone (Fountoulakis et al., J. Biochem 270:3958-3964(1995)).

[0087] Cancer Diagnosis and Prognosis

[0088] It is believed that certain tissues in mammals with cancer, inparticular acute myelogenous leukemias, express significantly alteredlevels of the HOIPS I protein and mRNA encoding the HOIPS I protein whencompared to a corresponding “standard” mammal, i.e., a mammal of thesame species not having the cancer. Further, it is believed thatenhanced levels of the HOIPS I protein can be detected in certain bodyfluids (e.g., sera, plasma, urine and spinal fluid) from mammals withcertain leukemias, e.g. acute myelogenous leukemia, when compared tosera from mammals of the same species not having the leukemia. Thus, theinvention provides a diagnostic method useful during myeloma diagnosis,which involves assaying the expression level of the gene encoding theHOIPS I protein in mammalian cells or body fluid and comparing the geneexpression level with a standard HOIPS I gene expression level, wherebyan increase in the gene expression level over the standard is indicativeof certain tumors.

[0089] Where a tumor diagnosis has already been made according toconventional methods, the present invention is useful as a prognosticindicator, whereby patients exhibiting enhanced HOIPS I gene expressionwill be predicted to experience a worse clinical outcome relative topatients expressing the gene at a lower level.

[0090] By “assaying the expression level of the gene encoding the HOIPSI protein” is intended qualitatively or quantitatively measuring orestimating the level of the HOIPS I protein or the level of the mRNAencoding the HOIPS I protein in a first biological sample eitherdirectly (e.g., by determining or estimating absolute protein level ormRNA level) or relatively (e.g., by comparing to the HOIPS I proteinlevel or mRNA level in a second biological sample).

[0091] Preferably, the HOIPS I protein level or mRNA level in the firstbiological sample is measured or estimated and compared to a standardHOIPS I protein level or mRNA level, the standard being taken from asecond biological sample obtained from an individual not having thecancer. As will be appreciated in the art, once a standard HOIPS Iprotein level or mRNA level is known, it can be used repeatedly as astandard for comparison.

[0092] By “biological sample” is intended any biological sample obtainedfrom an individual, cell line, tissue culture, or other source whichcontains HOIPS I protein or mRNA. Biological samples include mammalianbody fluids (such as sera, plasma, urine, synovial fluid and spinalfluid) which contain secreted mature HOIPS I protein, and hematopoietictissues including the spleen, tonsils, bone marrow, dendritic cells,fetal and adult brain macrophages, B cells, lymph nodes etc.

[0093] The present invention is useful for detecting cancer in mammals.In particular the invention is useful during diagnosis of the followingpathological cell proliferative neoplasias: acute myelogenous leukemiasincluding acute monocytic leukemia, acute myeloblastic leukemia, acutepromyelocytic leukemia, acute myelomonocytic leukemia, acuteerythroleukernia, acute megakaryocytic leukemia, and acuteundifferentiated leukemia, etc.; and chronic myelogenous leukemiasincluding chronic myelomonocytic leukemia, chronic granulocyticleukemia, etc. Preferred mammals include monkeys, apes, cats, dogs,cows, pigs, horses, rabbits and humans. Particularly preferred arehumans.

[0094] Total cellular RNA can be isolated from a biological sample usingthe single-step guanidinium-thiocyanate-phenol-chloroform methoddescribed in Chomczynski and Sacchi Anal. Biochem. 162:156-159 (1987).Levels of mRNA encoding the HOIPS I protein are then assayed using anyappropriate method. These include Northern blot analysis (Harada et al.,Cell 63:303-312 (1990)), S1 nuclease mapping (Fujita et al., Cell49:357-367 (1987)), the polymerase chain reaction (PCR), reversetranscription in combination with the polymerase chain reaction (RT-PCR)(Makino et al., Technique 2:295-301 (1990)), and reverse transcriptionin combination with the ligase chain reaction (RT-LCR).

[0095] Assaying HOIPS I protein levels in a biological sample can occurusing antibody-based techniques. For example, HOIPS I protein expressionin tissues can be studied with classical immunohistological methods(Jalkanen, M., et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M.,et al., J. Cell. Biol. 105:3087-3096 (1987)).

[0096] Other antibody-based methods useful for detecting HOIPS I proteingene expression include immunoassays, such as the enzyme linkedimmunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitablelabels are known in the art and include enzyme labels, such as, Glucoseoxidase, and radioisotopes, such as iodine (¹²⁵I, ¹²¹I), carbon (¹⁴C),salphee (³⁵S), tritium (³H), indium (¹¹²In), and technetium (^(99m)Tc),and fluorescent labels, such as fluorescein and rhodamine, and biotin.

[0097] Therapeutics

[0098] Pathological cell proliferative disorders are often associatedwith inappropriate activation of proto-oncogenes. (Gelmann, E. P. etal., “The Etiology of Acute Leukemia: Molecular Genetics and ViralOncology,” in Neoplastic Diseases of the Blood, Vol 1., Wiernik, P. H.et al. eds., 161-182 (1985)). Neoplasias are now believed to result fromthe qualitative alteration of a normal cellular gene product, or fromthe quantitative modification of gene expression by insertion into thechromosome of a viral sequence, by chromosomal translocation of a geneto a more actively transcribed region, or by some other mechanism.(Gelmann et al.) It is likely that mutated or altered expression ofspecific genes is involved in the pathogenesis of some leukemias.(Gelmann et al.) Indeed, the human counterparts of the oncogenesinvolved in some animal neoplasias have been amplified or translocatedin some cases of human leukemia and carcinoma. (Gelmann et al.).

[0099] For example, c-myc expression is highly amplified in thenon-lymphocytic leukemia cell line HL-60. When HL-60 cells arechemically induced to stop proliferation, the level of c-myc is found tobe downregulated. (WO 91/15580) However, it has been shown that exposureof HL-60 cells to a DNA construct that is complementary to the 5′ end ofc-myc or c-myb blocks translation of the corresponding mRNAs whichdownregulates expression of the c-myc or c-myb proteins and causesarrest of cell proliferation and differentiation of the treated cells.(WO 91/15580; Wickstrom et al., Proc. Natl. Acad. Sci. 85:1028 (1988);Anfossi et al., Proc. Natl. Acad. Sci. 86:3379 (1989)).

[0100] Accordingly, the present invention is directed to the utilizationof the HOIPS I gene and its product in gene therapy techniques to treatcell proliferative diseases in individuals. The term “gene therapy” ismeant to include the insertion of part of all of the HOIPS I gene, aHOIPS I DNA or RNA construct or HOIPS I gene product into a cell, groupof cells, tissue, pathological lesion, organ or organism for the purposeof modulating gene expression, and/or function of the gene product.

[0101] Thus, in one embodiment, the present invention provides a methodfor treating cell proliferative diseases, and in particular acute andchronic myelogenous leukemias, by inserting into an abnormallyproliferating cell which expresses the HOIPS I gene a synthetic DNA orRNA construct of the present invention, wherein said DNA or RNAconstruct represses said expression.

[0102] Another embodiment of the present invention provides a method oftreating cell-proliferative disorders in individuals comprisingadministration of one or more active gene copies of the HOIPS I gene toan abnormally proliferating cell or cells. In a preferred embodiment,the HOIPS I gene is a DNA construct comprising a recombinant expressionvector effective in expressing a DNA sequence encoding said HOIPS Igene. In another preferred embodiment of the present invention, the DNAconstruct encoding the HOIPS I gene is inserted into cells to be treatedutilizing a retrovirus vector. In a most preferred embodiment, theretroviral vector is defective and will not transform non-proliferatingcells.

[0103] By “repressing expression of the HOIPS I gene” is intended thesuppression of the transcription of the gene, the degradation of thegene transcript (pre-message RNA), the inhibition of splicing, thedestruction of the messenger RNA, the prevention of thepost-translational modifications of the protein, the destruction of theprotein, or the inhibition of the normal function of the protein.

[0104] In an especially preferred embodiment, suppression of HOIPS Igene expression in a cell is achieved by administering antisense RNA.Antisense RNAs are RNAs that are complimentary to all or part of themRNA of the HOIPS I gene. In general, overproduction of antisense RNAhas been shown to prevent translation of a given target RNA, therebyblocking the expression of the target gene product. (WO 91/15580).Accordingly, in order to block HOIPS I induced proliferation ordifferentiation of a cell, antisense RNAs can be introduced into theproliferating or differentiating cells.

[0105] The use of c-myc and c-myb antisense RNA constructs to inhibitthe growth of the non-lymphocytic leukemia cell line HL-60 and othercell lines was previously described. (Wickstrom et al. (1988); Anfossiet al. (1989)). These experiments were performed in vitro by incubatingcells with the oligoribonucleotide. A similar procedure for in vivo useis described in WO 91/15580. Briefly, a pair of oligonucleotides for agiven antisense RNA is produced as follows: A sequence complimentary tothe first 15 bases of the open reading frame is flanked by an EcoRI siteon the 5′ end and a HindIII site on the 3′ end. Next, the pair ofoligonucleotides is heated at 90° C. for one minute and then annealed in2× ligation buffer (20 mM TRIS HCl pH 7.5, 10 mM MgCl₂, 10 MMdithiothreitol (DTT) and 0.2 mM ATP) and then ligated to the EcoR1/HindIII site of the retroviral vector PMV7. (WO 91/15580).

[0106] It will be appreciated that conditions caused by a decrease inthe standard or normal level of HOIPS I activity in an individual, canbe treated by administration of HOIPS I protein. Thus, the inventionfurther provides a method of treating an individual in need of anincreased level of HOIPS I activity comprising administering to such anindividual a pharmaceutical composition comprising an effective amountof an isolated HOIPS I polypeptide of the invention, particularly amature form of the HOIPS I, effective to increase the HOIPS I activitylevel in such an individual.

[0107] As a general proposition, the total pharmaceutically effectiveamount of HOIPS I polypeptide administered parenterally per dose will bein the range of about 1 μg/kg/day to 10 mg/kg/day of patient bodyweight, although, as noted above, this will be subject to therapeuticdiscretion. More preferably, this dose is at least 0.01 mg/kg/day, andmost preferably for humans between about 0.01 and 1 mg/kg/day for thehormone. If given continuously, the HOIPS I polypeptide is typicallyadministered at a dose rate of about 1 μg/kg/hour to about 50μg/kg/hour, either by 1-4 injections per day or by continuoussubcutaneous infusions, for example, using a mini-pump. An intravenousbag solution may also be employed.

[0108] Pharmaceutical compositions containing the HOIPS I of theinvention may be administered orally, rectally, parenterally,intracistemally, intravaginally, intra peritoneally, topically (as bypowders, ointments, drops or transdernal patch), bucally, or as an oralor nasal spray. By “pharmaceutically acceptable carrier” is meant anon-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any type. The term “parenteral” asused herein refers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

[0109] For local administration to abnormally proliferating cells, theHOIPS I DNA or RNA constructs or genes may be administered by any methodknown to those of skill in the art including, but not limited totransfection, electroporation, microinjection of cells, or in vehiclessuch as liposomes, lipofectin, or as naked DNA or RNA. The DNA of thepresent invention may be delivered by known gene delivery systems suchas, but not limited to, retroviral vectors (Gilboa, J. Virology 44:845(1982); Hocke, Nature 320:275 (1986); Wilson, et al., Proc. Natl. Acad.Sci. U.SA. 85:3014), vaccinia virus system (Chakrabarty et al., Mol.Cell Biol. 5:3403 (1985) or other efficient DNA delivery systems (Yateset al., Nature 313:812 (1985)) known to those skilled in the art. Thesereferences are exemplary only and are hereby incorporated by reference.In order to specifically deliver or transfect cells which are abnormallyproliferating and spare non-dividing cells, it is preferable to utilizea retrovirus delivery system known to those of skill in the art. Sincehost DNA replication is required for retroviral DNA to integrate and theretrovirus will be unable to self replicate due to the lack of theretrovirus genes needed for its life cycle. Utilizing such a retroviraldelivery system for the HOIPS I gene and DNA constructs of the presentinvention will target said gene and constructs to abnormallyproliferating cells and will spare the non-dividing normal cells.

[0110] Administration of the HOIPS I gene, DNA or RNA constructs, orgene product useful in the methods of the present invention may be bytopical, parenteral, oral, intranasal, intravenous, intramuscular,subcutaneous, or any other suitable means.

[0111] The DNA constructs of the present invention may be delivereddirectly to cell proliferative disorder/disease sites in internalorgans, body cavities and the like by use of imaging devices used toguide an injecting needle directly to the disease site. The DNAconstructs of the present invention may also be administered to diseasesites at the time of surgical intervention.

[0112] The DNA dosage administered is dependent upon the age, clinicalstage and extent of the disease or genetic predisposition of theindividual, location, weight, kind of concurrent treatment, if any, andnature of the pathological or malignant cell proliferative disorder. Theeffective delivery system useful in the method of the present inventionmay be employed in such forms as capsules, tablets, liquid solutions,suspensions, or elixirs, for oral administration or sterile liquid formssuch as solutions, suspensions, or emulsions. Any inert carrier ispreferably used, such as saline, or phosphate-buffered saline, or anysuch carrier in which the compounds used in the method of the presentinvention have suitable solubility properties.

[0113] By “cell proliferative disease” is meant any human or animaldisease or disorder, affecting any one or any combination of organs,cavities, or body parts, which is characterized by single or multiplelocal abnormal proliferations of cells, groups of cells, or tissues,whether benign or malignant.

[0114] Any amount of the DNA or RNA constructs of the present inventionmay be administered as long as it has a biologically inhibiting effecton the proliferation of the treated cells. Moreover, it is possible toadminister more than one of the DNA or RNA constructs of the presentinvention simultaneously to the same site. By “biologically inhibiting”is meant partial or total growth inhibition as well as decreases in therate of proliferation or growth of the cells. The biologicallyinhibitory dose may be determined by assessing the effects of the sampleDNA or RNA constructs of the present invention on target malignant orabnormally proliferating cell growth in tissue culture, tumor growth inanimals and cell cultures, or any other method known to one of ordinaryskill in the art. The present invention is further directed toantibody-based therapies which involve administering an anti-HOIPS Iantibody to a mammalian, preferably human, patient for treating one ormore of the above-described disorders. Methods for producing anti-HOIPSI polyclonal and monoclonal antibodies are described in detail supra.Such antibodies may be provided in pharmaceutically acceptablecompositions as known in the art or as described herein.

[0115] A summary of the ways in which the antibodies of the presentinvention may be used therapeutically includes binding HOIPS I locallyor systemically in the body or by direct cytotoxicity of the antibody,e.g as mediated by complement (CDC) or by effector cells (ADCC). Some ofthese approaches are described in more detail below. Armed with theteachings provided herein, one of ordinary skill in the art will knowhow to use the antibodies of the present invention for diagnostic,monitoring or therapeutic purposes without undue experimentation.

[0116] The pharmaceutical compositions of the present invention may beadministered by any means that achieve their intended purpose. Amountsand regimens for the administration of antibodies, their fragments orderivatives can be determined readily by those with ordinary skill inthe clinical art of treating cell proliferative diseases.

[0117] For example, administration may be by parenteral, subcutaneous,intravenous, intramuscular, intraperitoneal, transdermal, or buccalroutes. Alternatively, or concurrently, administration may be by theoral route. The dosage administered will be dependent upon the age,health and weight of the recipient, kind of concurrent treatment, ifany, frequency of treatment, and the nature of the desired effect.

[0118] Compositions within the scope of this invention include allcompositions wherein the antibody, fragment or derivative is containedin an amount effective to achieve its intended purpose. While individualneeds vary, determination of optimal ranges of effective amounts of eachcomponent is within the skill of the art. The effective dose is afunction of the individual chimeric or monoclonal antibody, the presenceand nature of a conjugated therapeutic agent (see below), the patientand his clinical status, and can vary from about 10 μg/kg body weight toabout 5000 mg/kg body weight. The preferred dosages comprise 0.1-500mg/kg body wt.

[0119] In addition to the pharmacologically active compounds, the newpharmaceutical compositions may contain suitable pharmaceuticallyacceptable carriers comprising excipients and auxiliaries whichfacilitate processing of the active compounds into preparations whichcan be used pharmaceutically. Preferably, the preparations contain fromabout 0.01 to 99 percent, preferably from about 20-75 percent of activecompound(s), together with the excipient.

[0120] Similarly, preparations of an anti-HOIPS I antibody, or antigenbinding fragment thereof, of the present invention for parenteraladministration, such as in detectably labeled form for imaging or in afree or conjugated form for therapy, include sterile aqueous ornon-aqueous solutions, suspensions, or emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oil, such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia, parenteral vehicles including sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers, suchas those based on Ringer's dextrose, and the like. Preservatives andother additives may also be present, such as, for example,antimicrobials, antioxidants, chelating agents, and inert gases and thelike. See, generally, Remington's Pharmaceutical Science, 16th ed., MackPublishing Co., Easton, Pa., 1980.

[0121] In particular, the antibodies, fragments and derivatives of thepresent invention are useful for treating a subject having or developingHOIPS I related cell proliferative and/or differentiation disorders asdescribed herein. Such treatment comprises administering a single ormultiple doses of the antibody, or a fragment, derivative, or aconjugate thereof.

[0122] The antibodies of this invention may be advantageously utilizedin combination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors, etc., which serve toincrease the number or activity of effector cells which interact withthe antibodies.

[0123] It is preferred to use high affinity and/or potent in vivo HOIPSI inhibiting and/or neutralizing antibodies, fragments or regionsthereof, for both HOIPS I immunoassays and therapy of HOIPS I relateddisorders. Such antibodies, fragments, or regions, will preferably havean affinity for human HOIPS I, expressed as Ka, of at least 10⁸ M⁻¹,more preferably, at least 10⁹ M⁻¹, such as 5×10⁸ M⁻¹, 8×10⁸ M⁻¹, 2×10⁹M⁻¹, 4×10⁹ M⁻¹, 6×10⁹M⁻¹, 8×10⁹ M⁻¹.

[0124] Preferred for human therapeutic use are high affinity murine andmurine/human or human/human chimeric antibodies, and fragments, regions,and derivatives thereof having potent in vivo HOIPS I inhibiting and/orneutralizing activity, according to the present invention, e.g., thatblock HOIPS I activity.

[0125] Chromosome Assays

[0126] The nucleic acid molecules of the present invention are alsovaluable for chromosome identification. The sequence is specificallytargeted to and can hybridize with a particular location on anindividual human chromosome. The mapping of DNAs to chromosomesaccording to the present invention is an important first step incorrelating those sequences with genes associated with disease.

[0127] In certain preferred embodiments in this regard, the cDNA hereindisclosed is used to clone genomic DNA of a HOIPS I protein gene. Thiscan be accomplished using a variety of well known techniques andlibraries, which generally are available commercially. The genomic DNAthen is used for in situ chromosome mapping using well known techniquesfor this purpose.

[0128] In addition, in some cases, sequences can be mapped tochromosomes by preparing PCR primers (preferably 15-25 bp) from thecDNA. Computer analysis of the 3′ untranslated region of the gene isused to rapidly select primers that do not span more than one exon inthe genomic DNA, thus complicating the amplification process. Theseprimers are then used for PCR screening of somatic cell hybridscontaining individual human chromosomes.

[0129] Fluorescence in situ hybridization (“FISH”) of a cDNA clone to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with probesfrom cDNA as short as 50 or 60 bp. For a review of this technique, seeVerma et al., Human Chromosomes: A Manual Of Basic Techniques, PergamonPress, New York (1988).

[0130] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data. Such data are found, for example, inV. McKusick, Mendelian Inheritance In Man, available on-line throughJohns Hopkins University, Welch Medical Library. The relationshipbetween genes and diseases that have been mapped to the same chromosomalregion are then identified through linkage analysis (coinheritance ofphysically adjacent genes).

[0131] Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

[0132] Thus, in one embodiment of the present invention these techniquescan be used to identify individuals who are predisposed to cellproliferative diseases. Specifically, the present inventions can be usedto screen chromosomal DNA of an individual to determine the presence orabsence of active alleles of the HOIPS I gene. Those having only oneactive allele of the HOIPS I gene are predicted to be predisposed tocell proliferative disorders.

[0133] Having generally described the invention, the same will be morereadily understood by reference to the following examples, which areprovided by way of illustration and are not intended as limiting.

EXAMPLES Example 1 Expression and Purfication of HOIPS I in E. coli

[0134] The DNA sequence encoding the mature HOIPS I protein in thedeposited cDNA clone is amplified using PCR oligonucleotide primersspecific to the amino terminal sequences of the HOIPS I protein and tovector sequences 3′ to the gene. Additional nucleotides containingrestriction sites to facilitate cloning are added to the 5′ and 3′sequences respectively.

[0135] The 5′ oligonucleotide primer has the sequence:

[0136] 5′ GACTCCATGGGCGGCGGTGGGAAAGCCTG 3′ (SEQ ID NO:4) containing theunderlined NcoI restriction site, which encodes 20 nucleotides of theHOIPS I protein coding sequence in FIG. 1 (SEQ ID NO: 1) beginningimmediately after the signal peptide.

[0137] The 3′ primer has the sequence:

[0138] 5′ GACTAGATCTGGAGCACATGATAGTAGCAT 3′ (SEQ ID NO:5) containing theunderlined BglII restriction site followed by 20 nucleotidescomplementary to the last 20 nucleotides of the HOIPS I protein codingsequence in FIG. 1.

[0139] The restriction sites are convenient to restriction enzyme sitesin the bacterial expression vector nQE60, which are used for bacterialexpression in these examples. (Qiagen, Inc. 9259 Eton Avenue,Chatsworth, Calif., 91311). nQE60 encodes ampicillin antibioticresistance (“Ampr”) and contains a bacterial origin of replication(“ori”), an IPTG inducible promoter, a ribosome binding site (“RBS”), a6-His tag and restriction enzyme sites.

[0140] The amplified HOIPS I DNA and the vector nQE60 both are digestedwith NcoI and BglII and the digested DNAs are then ligated together.Insertion of the HOIPS I protein DNA into the restricted nQE60 vectorplaces the HOIPS I protein coding region downstream of and operablylinked to the vector's IPTG-inducible promoter and in-frame with aninitiating AUG appropriately positioned for translation of HOIPS Iprotein.

[0141] The ligation mixture is transformed into competent E. coli cellsusing standard procedures. Such procedures are described in Sambrook etal., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989). E. coli strainM15/rep4, containing multiple copies of the plasmid pREP4, whichexpresses lac repressor and confers kanamycin resistance (“Kan^(r)”), isused in carrying out the illustrative example described herein. Thisstrain, which is only one of many that are suitable for expressing HOIPSI protein, is available commercially from Qiagen.

[0142] Transformants are identified by their ability to grow on LBplates in the presence of ampicillin and kanamycin. Plasmid DNA isisolated from resistant colonies and the identity of the cloned DNAconfirmed by restriction analysis.

[0143] Clones containing the desired constructs are grown overnight(“O/N”) in liquid culture in LB media supplemented with both ampicillin(100 μg/ml) and kanamycin (25 μg/ml).

[0144] The O/N culture is used to inoculate a large culture, at adilution of approximately 1:100 to 1:250. The cells are grown to anoptical density at 600 nm (“OD600”) of between 0.4 and 0.6.Isopropyl-B-D-thiogalactopyranoside (“IPTG”) is then added to a finalconcentration of 1 mM to induce transcription from lac repressorsensitive promoters, by inactivating the lacI repressor. Cellssubsequently are incubated further for 3 to 4 hours. Cells then areharvested by centrifugation and disrupted, by standard methods.Inclusion bodies are purified from the disrupted cells using routinecollection techniques, and protein is solubilized from the inclusionbodies into 8M urea. The 8M urea solution containing the solubilizedprotein is passed over a PD-10 column in 2× phosphate-buffered saline(“PBS”), thereby removing the urea, exchanging the buffer and refoldingthe protein. The protein is purified by a further step of chromatographyto remove endotoxin. Then, it is sterile filtered. The sterile filteredprotein preparation is stored in 2× PBS at a concentration of 95 μ/ml.

Example 2 Cloning and Expression of HOIPS I protein in a BaculovirusExpression System

[0145] In this illustrative example, the plasmid shuttle vector pA2 isused to insert the cloned DNA encoding the complete protein, includingits naturally associated secretary signal (leader) sequence, into abaculovirus to express the mature HOIPS I protein, using standardmethods as described in Summers et al., A Manual of Methods forBaculovirus Vectors and Insect Cell Culture Procedures, TexasAgricultural Experimental Station Bulletin No 1555 (1987). Thisexpression vector contains the strong polyhedrin promoter of theAutographa californica nuclear polyhedrosis virus (AcMNPV) followed byconvenient restriction sites such as BamHI and Asp718. Thepolyadenylation site of the simian virus 40 (“SV40”) is used forefficient polyadenylation. For easy selection of recombinant virus, theplasmid contains the beta-galactosidase gene from E. coli under controlof a weak Drosophila promoter in the same orientation, followed by thepolyadenylation signal of the polyhedrin gene. The inserted genes areflanked on both sides by viral sequences for cell-mediated homologousrecombination with wild-type viral DNA to generate viable virus thatexpress the cloned polynucleotide.

[0146] Many other baculovirus vectors could be used in place of thevector above, such as pAc373, pVL941 and pAcIM1, as one skilled in theart would readily appreciate, as long as the construct providesappropriately located signals for transcription, translation, secretionand the like, including a signal peptide and an in-frame AUG asrequired. Such vectors are described, for instance, in Luckow et al.,Virology 170:31-39.

[0147] The cDNA sequence encoding the full length HOIPS I protein in thedeposited clone, including the AUG initiation codon and the naturallyassociated leader sequence shown in FIG. 1 (SEQ ID NO:2), is amplifiedusing PCR oligonucleotide primers corresponding to the 5′ and 3′sequences of the gene. The 5′ primer has the sequence 5′ GAC TGGATCCGCCATC ATG AAG GGT TTC ACA GCC AC 3′ (SEQ ID NO:6) containing theunderlined BamHI restriction enzyme site, an efficient signal forinitiation of translation in eukaryotic cells, as described by Kozak,M., J. Mol. Biol. 196:947-950 (1987), followed by 20 bases of thesequence of the complete HOIPS I protein shown in FIG. 1, beginning withthe AUG initiation codon. The 3′ primer has the sequence 5′GACTGGTACCAG- CAGCTGCACTCTTTGGG 3′ (SEQ ID NO: 7) containing theunderlined, Asp718 restriction site followed by 19 nucleotidescomplementary to the 3′ noncoding sequence in FIG. 1.

[0148] The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with BamHI and Asp718 and againis purified on a 1% agarose gel. This fragment is designated herein“F1”.

[0149] The plasmid is digested with the restriction enzymes BamHI andAsp718 and optionally, can be dephosphorylated using calf intestinalphosphatase, using routine procedures known in the art. The DNA is thenisolated from a 1% agarose gel using a commercially available kit(“Geneclean” BIO 101 Inc., La Jolla, Calif.). This vector DNA isdesignated herein “V1”.

[0150] Fragment F1 and the dephosphorylated plasmid V1 are ligatedtogether with T4 DNA ligase. E. coli HB101 or other suitable E. colihosts such as XL-1 Blue (Stratagene Cloning Systems, La Jolla, Calif.)cells are transformed with the ligation mixture and spread on cultureplates. Bacteria are identified that contain the plasmid with the humanHOIPS I gene using the PCR method, in which one of the primers that isused to amplify the gene and the second primer is from well within thevector so that only those bacterial colonies containing the HOIPS I genefragment will show amplification of the DNA. The sequence of the clonedfragment is confirmed by DNA sequencing. This plasmid is designatedherein pBacHOIPS I.

[0151] Five μg of the plasmid pBacHOIPS I is co-transfected with 1.0 μgof a commercially available linearized baculovirus DNA (“BaculoGold™baculovirus DNA”, Pharmingen, San Diego, Calif.), using the lipofectionmethod described by Felgner et al., Proc. Natl. Acad. Sci. USA84:7413-7417 (1987). 1 μg of BaculoGold™ virus DNA and 5 μg of theplasmid pBacHOIPS I are mixed in a sterile well of a microtiter platecontaining 50 μl of serum-free Grace's medium (Life Technologies Inc.,Gaithersburg, Md.). Afterwards, 10 μl Lipofectin plus 90 μl Grace'smedium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added drop-wise to Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plate is rocked back and forth tomix the newly added solution. The plate is then incubated for 5 hours at27° C. After 5 hours the transfection solution is removed from the plateand 1 ml of Grace's insect medium supplemented with 10% fetal calf serumis added. The plate is put back into an incubator and cultivation iscontinued at 27° C. for four days.

[0152] After four days the supernatant is collected and a plaque assayis performed, as described by Summers and Smith, supra. An agarose gelwith “Blue Gal” (Life Technologies Inc., Gaithersburg) is used to alloweasy identification and isolation of gal-expressing clones, whichproduce blue-stained plaques. (A detailed description of a “plaqueassay” of this type can also be found in the user's guide for insectcell culture and baculovirology distributed by Life Technologies Inc.,Gaithersburg, page 9-10). After appropriate incubation, blue stainedplaques are picked with the tip of a micropipettor (e.g., Eppendorf).The agar containing the recombinant viruses is then resuspended in amicrocentrifuge tube containing 200 μl of Grace's medium and thesuspension containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4° C. Therecombinant virus is called V-HOIPS I.

[0153] To verify the expression of the gene, Sf9 cells are grown inGrace's medium supplemented with 10% heat-inactivated FBS. The cells areinfected with the recombinant baculovirus V-HOIPS I at a multiplicity ofinfection (“MOI”) of about 2. Six hours later the medium is removed andis replaced with SF900 II medium minus methionine and cysteine(available from Life Technologies Inc., Rockville, Md.). If radiolabeledproteins are desired, 42 hours later, 5 μCi of ³⁵S-methionine and 5 μCi³⁵S-cysteine (available from Amersham) are added. The cells are furtherincubated for 16 hours and then they are harvested by centrifugation.The proteins in the supernatant as well as the intracellular proteinsare analyzed by SDS-PAGE followed by autoradiography (if radiolabeled).Microsequencing of the amino acid sequence of the amino terminus ofpurified protein may be used to determine the amino terminal sequence ofthe mature protein and thus the cleavage point and length of thesecretory signal peptide.

Example 3 Cloning and Expression in Mammalian Cells

[0154] A typical mammalian expression vector contains the promoterelement, which mediates the initiation of transcription of MRNA, theprotein coding sequence, and signals required for the termination oftranscription and polyadenylation of the transcript. Additional elementsinclude enhancers, Kozak sequences and intervening sequences flanked bydonor and acceptor sites for RNA splicing. Highly efficienttranscription can be achieved with the early and late promoters fromSV40, the long terminal repeats (LTRS) from Retroviruses, e.g., RSV,HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).However, cellular elements can also be used (e.g., the human actinpromoter). Suitable expression vectors for use in practicing the presentinvention include, for example, vectors such as PSVL and PMSG(Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC37146) and pBC12MI (ATCC 67109). Mammalian host cells that could be usedinclude, human Hela 293, H9 and Jurkat cells, mouse NIH3T3 and C127cells, Cos 1, Cos 7 and CV 1, quail QC1-3 cells, mouse L cells andChinese hamster ovary (CHO) cells.

[0155] Alternatively, the gene can be expressed in stable cell linesthat contain the gene integrated into a chromosome. The co-transfectionwith a selectable marker such as dhfr, gpt, neomycin or hygromycinallows the identification and isolation of the transfected cells.

[0156] The transfected gene can also be amplified to express largeamounts of the encoded protein. The DHFR (dihydrofolate reductase)marker is useful to develop cell lines that carry several hundred oreven several thousand copies of the gene of interest. Another usefulselection marker is the enzyme glutarnine synthase (GS) (Murphy et al.,Biochem J. 227:277-279 (1991); Bebbington et al., Bio/Technology10:169-175 (1992)). Using these markers, the mammalian cells are grownin selective medium and the cells with the highest resistance areselected. These cell lines contain the amplified gene(s) integrated intoa chromosome. Chinese hamster ovary (CHO) and NSO cells are often usedfor the production of proteins.

[0157] The expression vectors pC1 and pC4 contain the strong promoter(LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular and CellularBiology, 438-447 (March, 1985)) plus a fragment of the CMV-enhancer(Boshart et al., Cell 41:521-530 (1985)). Multiple cloning sites, e.g.,with the restriction enzyme cleavage sites BamHI, XbaI and Asp718,facilitate the cloning of the gene of interest. The vectors contain inaddition the 3′ intron, the polyadenylation and termination signal ofthe rat preproinsulin gene.

Example 3(a) Cloning and Expression in COS Cells

[0158] The expression plasmid, pHOIPS I HA, is made by cloning a cDNAencoding HOIPS I into the expression vector pcDNAI/Amp or pcDNAIII(which can be obtained from Invitrogen, Inc.).

[0159] The expression vector pcDNAI/amp contains: (1) an E. coli originof replication effective for propagation in E. coli and otherprokaryotic cells; (2) an ampicillin resistance gene for selection ofplasrnid-containing prokaryotic cells; (3) an SV40 origin of replicationfor propagation in eukaryotic cells; (4) a CMV promoter, a polylinker,an SV40 intron; (5) several codons encoding a hemagglutinin fragment(i.e., an “HA” tag to facilitate purification) followed by a terminationcodon and polyadenylation signal arranged so that a eDNA can beconveniently placed under expression control of the CMV promoter andoperably linked to the SV40 intron and the polyadenylation signal bymeans of restriction sites in the polylinker. The HA tag corresponds toan epitope derived from the influenza hemagglutinin protein described byWilson et al., Cell 37:767 (1984). The fusion of the HA tag to thetarget protein allows easy detection and recovery of the recombinantprotein with an antibody that recognizes the HA epitope. pcDNAIIIcontains, in addition, the selectable neomycin marker.

[0160] A DNA fragment encoding the HOIPS I is cloned into the polylinkerregion of the vector so that recombinant protein expression is directedby the CMV promoter. The plasmid construction strategy is as follows.The HOIPS I cDNA of the deposited clone is amplified using primers thatcontain convenient restriction sites, much as described above forconstruction of vectors for expression of HOIPS I in E. coli. Suitableprimers include the following, which are used in this example. The 5′primer, containing the underlined HindIII site, an AUG start codon and 6codons of the 5′ coding region of the complete HOPS I polypeptide hasthe following sequence:

[0161] 5′ AGCTAAGCTTCCGCCACCATGAAGGGTTTCACAGCC 3′ (SEQ ID NO:8). The 3′primer, containing the underlined XhoI site, a stop codon, and 22 bp of3′ coding sequence has the following sequence:5′CAGTCTCGAGTTAAGCGTAGTCTGGGACGTCGTATGGGTAGGAGCA (SEQ ID NO:9).CATGATAGTAGCATTG 3′

[0162] The PCR amplified DNA fragment and the vector, pcDNAI/Amp, aredigested with HindIII and XhoI and then ligated. The ligation mixture istransformed into E. coli strain SURE (available from Stratagene CloningSystems, 11099 North Torrey Pines Road, La Jolla, Calif. 92037), and thetransformed culture is plated on ampicillin media plates which then areincubated to allow growth of ampicillin resistant colonies. Plasmid DNAis isolated from resistant colonies and examined by restriction analysisor other means for the presence of the HOIPS I-encoding fragment.

[0163] For expression of recombinant HOIPS I, COS cells are transfectedwith an expression vector, as described above, using DEAE-DEXTRAN, asdescribed, for instance, in Sambrook et al., Molecular Cloning: aLaboratory Manual, Cold Spring Laboratory Press, Cold Spring Harbor,N.Y. (1989). Cells are incubated under conditions for expression ofHOIPS I by the vector.

[0164] Expression of the HOIPS I-HA fusion protein is detected byradiolabeling and immunoprecipitation, using methods described in, forexample Harlow et al., Antibodies: A Laboratory Manual, 2nd Ed.; ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988). To thisend, two days after transfection, the cells are labeled by incubation inmedia containing ³⁵S-cysteine for 8 hours. The cells and the media arecollected, and the cells are washed and lysed with detergent-containingRIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM TRIS, pH7.5, as described by Wilson et al. cited above. Proteins areprecipitated from the cell lysate and from the culture media using anHA-specific monoclonal antibody. The precipitated proteins then areanalyzed by SDS-PAGE and autoradiography. An expression product of theexpected size is seen in the cell lysate, which is not seen in negativecontrols.

Example 3 (b) Cloning and Expression in CHO Cells

[0165] The vector pC4 is used for the expression of HOIPS I protein.Plasmid pC4 is a derivative of the plasmid pSV2-dhfr (ATCC Accession No.37146). The plasmid contains the mouse DHFR gene under control of theSV40 early promoter. Chinese hamster ovary- or other cells lackingdihydrofolate activity that are transfected with these plasmids can beselected by growing the cells in a selective medium (alpha minus MEM,Life Technologies) supplemented with the chemotherapeutic agentmethotrexate. The amplification of the DHFR genes in cells resistant tomethotrexate (MTX) has been well documented (see, e.g., Alt, F. W.,Kellems, R. M., Bertino, J. R., and Schimke, R. T., 1978, J. Biol. Chem.253:1357-1370, Hamlin, J. L. and Ma, C. 1990, Biochem. et Biophys. Acta,1097:107-143, Page, M. J. and Sydenham, M. A. 1991, Biotechnology9:64-68). Cells grown in increasing concentrations of MTX developresistance to the drug by overproducing the target enzyme, DHFR, as aresult of amplification of the DHFR gene. If a second gene is linked tothe DHFR gene, it is usually co-amplified and over-expressed. It isknown in the art that this approach may be used to develop cell linescarrying more than 1,000 copies of the amplified gene(s). Subsequently,when the methotrexate is withdrawn, cell lines are obtained whichcontain the amplified gene integrated into one or more chromosome(s) ofthe host cell.

[0166] Plasmid pC4 contains for expressing the gene of interest thestrong promoter of the long terminal repeat (LTR) of the Rous SarcomaVirus (Cullen, et al., Molecular and Cellular Biology, March1985:438-447) plus a fragment isolated from the enhancer of theimmediate early gene of human cytomegalovirus (CMV) (Boshart et al.,Cell 41:521-530 (1985)). Downstream of the promoter are BamHI, XbaI, andAsp718 restriction enzyme cleavage sites that allow integration of thegene. Behind these cloning sites the plasmid contains the 3′ intron andpolyadenylation site of the rat preproinsulin gene. Other highefficiency promoters can also be used for the expression, e.g., thehuman β-actin promoter, the SV40 early or late promoters or the longterminal repeats from other retroviruses, e.g., HIV and HTLVI.Clontech's Tet-Off and Tet-On gene expression systems and similarsystems can be used to express the HOIPS I in a regulated way inmammalian cells (Gossen, M., & Bujard, H. 1992, Proc. Natl. Acad. Sci.USA 89: 5547-5551). For the polyadenylation of the mRNA other signals,e.g., from the human growth hormone or globin genes can be used as well.Stable cell lines carrying a gene of interest integrated into thechromosomes can also be selected upon co-transfection with a selectablemarker such as gpt, G418 or hygromycin. It is advantageous to use morethan one selectable marker in the beginning, e.g., G418 plusmethotrexate.

[0167] The plasmid pC4 is digested with the restriction enzyme BamHI andthen dephosphorylated using calf intestinal phosphatase by proceduresknown in the art. The vector is then isolated from a 1% agarose gel.

[0168] The DNA sequence encoding the complete HOIPS I protein includingits leader sequence is amplified using PCR oligonucleotide primerscorresponding to the 5′ and 3′ sequences of the gene. The 5′ primer hasthe sequence 5′ GACTGGATCCGCCATCATGAAGGGTTTCACAGCCAC 3′ (SEQ ID NO:6)containing the underlined BamHI restriction enzyme site followed by anefficient signal for initiation of translation in eukaryotes, asdescribed by Kozak, M., J. Mol. Biol. 196:947-950 (1987), and 20 basesof the coding sequence of HOIPS I shown in FIG. 1 (SEQ ID NO: 1). The 3′primer has the sequence 5′ GACTGGTACCAGCAGCTGCACTCTTTGGG 3′ (SEQ IDNO:10) containing the underlined Asp718 restriction site followed by 19nucleotides complementary to the non-translated region of the HOIPS Igene shown in FIG. 1 (SEQ ID NO: 1).

[0169] The amplified fragment is digested with the endonucleases BamHIand Asp718 and then purified again on a 1% agarose gel. The isolatedfragment and the dephosphorylated vector are then ligated with T4 DNAligase. E. coli HB 101 or XL-1 Blue cells are then transformed andbacteria are identified that contain the fragment inserted into plasmidpC4 using, for instance, restriction enzyme analysis.

[0170] Chinese hamster ovary cells lacking an active DHFR gene are usedfor transfection. 5 μg of the expression plasmid pC4 is cotransfectedwith 0.5 μg of the plasmid pSV2-neo using lipofectin (Felgner etal.,supra). The plasmid pSV2-neo contains a dominant selectable marker, theneo gene from Tn5 encoding an enzyme that confers resistance to a groupof antibiotics including G418. The cells are seeded in alpha minus MEMsupplemented with 1 mg/ml G418. After 2 days, the cells are trypsinizedand seeded in hybridoma cloning plates (Greiner, Germany) in alpha minusMEM supplemented with 10, 25, or 50 ng/mil of methotrexate plus 1 mg/mlG418. After about 10-14 days single clones are trypsinized and thenseeded in 6-well petri dishes or 10 ml flasks using differentconcentrations of methotrexate (50 nM, 100 nM, 200 n, 400 rM, 800 nM).Clones growing at the highest concentrations of methotrexate are thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (1 μM, 2 μM, 5 μM, 10 mM, 20 mM). The same procedure isrepeated until clones are obtained which grow at a concentration of100-200 μM. Expression of the desired gene product is analyzed, forinstance, by SDS-PAGE and Western blot or by reverse phase HPLCanalysis.

Example 4 Tissue Distribution of HOIPS I mRNA Expression

[0171] Northern blot analysis is carried out to examine HOIPS I geneexpression in human tissues, using methods described by, among others,Sambrook et al., cited above. A cDNA probe containing the entirenucleotide sequence of the HOIPS I protein (SEQ iID NO: 1) is labeledwith ³²P using the rediprime™ DNA labeling system (Amersham LifeScience), according to manufacturer's instructions. After labeling, theprobe is purified using a CHROMA SPIN-100™ column (ClontechLaboratories, Inc.), according to manufacturer's protocol numberPT1200-1. The purified labeled probe is then used to examine varioushuman tissues for HOIPS I mRNA.

[0172] Multiple Tissue Northern (MTN) blots containing various humantissues (H) or human immune system tissues (IM) are obtained fromClontech and are examined with the labeled probe using ExpressHyb™hybridization solution (Clontech) according to manufacturer's protocolnumber PT 1190-1. Following hybridization and washing, the blots aremounted and exposed to film at −70° C. overnight, and films developedaccording to standard procedures.

[0173] The HOIPS I gene has been found to be expressed in hematopoetictissues including: spleen, tonsils, bone marrow, dendritic cells, fetaland adult brain macrophages, B cells, lymph nodes etc.

[0174] It will be clear that the invention may be practiced otherwisethan as particularly described in the foregoing description andexamples.

[0175] Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, are withinthe scope of the appended claims.

[0176] The entire disclosure of all publications (including patents,patent applications, journal articles, laboratory manuals, books, orother documents) cited herein are hereby incorporated by reference.

1 17 860 base pairs nucleic acid double linear cDNA CDS 20..505sig_peptide 20..79 mat_peptide 80..505 1 TCCCATACAG GCCCCCACC ATG AAGGGT TTC ACA GCC ACT CTC TTC CTC TGG 52 Met Lys Gly Phe Thr Ala Thr LeuPhe Leu Trp -20 -15 -10 ACT CTG ATT TTT CCC AGC TGC AGT GGA GGC GGC GGTGGG AAA GCC TGG 100 Thr Leu Ile Phe Pro Ser Cys Ser Gly Gly Gly Gly GlyLys Ala Trp -5 1 5 CCC ACA CAC GTG GTC TGT AGC GAC AGC GGC TTG GAA GTGCTC TAC CAG 148 Pro Thr His Val Val Cys Ser Asp Ser Gly Leu Glu Val LeuTyr Gln 10 15 20 AGT TGC GAT CCA TTA CAA GAT TTT GGC TTT TCT GTT GAA AAGTGT TCC 196 Ser Cys Asp Pro Leu Gln Asp Phe Gly Phe Ser Val Glu Lys CysSer 25 30 35 AAG CAA TTA AAA TCA AAT ATC AAC ATT AGA TTT GGA ATT ATT CTGAGA 244 Lys Gln Leu Lys Ser Asn Ile Asn Ile Arg Phe Gly Ile Ile Leu Arg40 45 50 55 GAG GAC ATC AAA GAG CTT TTT CTT GAC CTA GCT CTC ATG TCT CAAGGC 292 Glu Asp Ile Lys Glu Leu Phe Leu Asp Leu Ala Leu Met Ser Gln Gly60 65 70 TCA TCT GTT TTG AAT TTC TCC TAT CCC ATC TGT GAG GCG GCT CTG CCC340 Ser Ser Val Leu Asn Phe Ser Tyr Pro Ile Cys Glu Ala Ala Leu Pro 7580 85 AAG TTT TCT TTC TGT GGA AGA AGG AAA GGA GAG CAG ATT TAC TAT GCT388 Lys Phe Ser Phe Cys Gly Arg Arg Lys Gly Glu Gln Ile Tyr Tyr Ala 9095 100 GGG CCT GTC AAT AAT CCT GAA TTT ACT ATT CCT CAG GGA GAA TAC CAG436 Gly Pro Val Asn Asn Pro Glu Phe Thr Ile Pro Gln Gly Glu Tyr Gln 105110 115 GTT TTG CTG GAA CTG TAC ACT GAA AAA CGG TCC ACC GTG GCC TGT GCC484 Val Leu Leu Glu Leu Tyr Thr Glu Lys Arg Ser Thr Val Ala Cys Ala 120125 130 135 AAT GCT ACT ATC ATG TGC TCC TGACTGTGGC CTGTAGCAAA AATCACAGCC535 Asn Ala Thr Ile Met Cys Ser 140 AGCTGCATCT CGTGGGACCT CCAAGCTCCTCTGACTGAAC CTACTGTGGG AGGAGAAGCA 595 GCTGATGACA GAGAGAGGCT CTACAAAGAAGCGCCCCCAA AGAGTGCAGC TGCTAATTTT 655 AGTCCCAGGA CCAGACATCC CCAGACTCCACAGATGTAAT GAAGTCCCCG AATGTATCTG 715 TTTCTAAGGA GCCTCTTGGC AGTCCTTAAGCAGTCTTGAG GGTCCATCCT TTTTCTCTAA 775 TTGGTCGCCT CCCACCAGAC TCACCTGCTTTTCAACTTTT TAGGAGTGCT TCCTCACAGT 835 TACCAAGAAA TAAAGAAAGC TGGCC 860 162amino acids amino acid linear protein 2 Met Lys Gly Phe Thr Ala Thr LeuPhe Leu Trp Thr Leu Ile Phe Pro -20 -15 -10 -5 Ser Cys Ser Gly Gly GlyGly Gly Lys Ala Trp Pro Thr His Val Val 1 5 10 Cys Ser Asp Ser Gly LeuGlu Val Leu Tyr Gln Ser Cys Asp Pro Leu 15 20 25 Gln Asp Phe Gly Phe SerVal Glu Lys Cys Ser Lys Gln Leu Lys Ser 30 35 40 Asn Ile Asn Ile Arg PheGly Ile Ile Leu Arg Glu Asp Ile Lys Glu 45 50 55 60 Leu Phe Leu Asp LeuAla Leu Met Ser Gln Gly Ser Ser Val Leu Asn 65 70 75 Phe Ser Tyr Pro IleCys Glu Ala Ala Leu Pro Lys Phe Ser Phe Cys 80 85 90 Gly Arg Arg Lys GlyGlu Gln Ile Tyr Tyr Ala Gly Pro Val Asn Asn 95 100 105 Pro Glu Phe ThrIle Pro Gln Gly Glu Tyr Gln Val Leu Leu Glu Leu 110 115 120 Tyr Thr GluLys Arg Ser Thr Val Ala Cys Ala Asn Ala Thr Ile Met 125 130 135 140 CysSer 133 amino acids amino acid Not Relevant Not Relevant protein 3 TrpPro Thr His Thr Val Cys Lys Glu Glu Asn Leu Glu Ile Tyr Tyr 1 5 10 15Lys Ser Cys Asp Pro Gln Gln Asp Phe Ala Phe Ser Ile Asp Arg Cys 20 25 30Ser Asp Val Thr Thr His Thr Phe Asp Ile Arg Ala Ala Met Val Leu 35 40 45Arg Gln Ser Ile Lys Glu Leu Tyr Ala Lys Val Asp Leu Ile Ile Asn 50 55 60Gly Lys Thr Val Leu Ser Tyr Ser Glu Thr Leu Cys Gly Pro Gly Leu 65 70 7580 Ser Lys Leu Ile Phe Cys Gly Lys Lys Lys Gly Glu His Leu Tyr Tyr 85 9095 Glu Gly Pro Ile Thr Leu Gly Ile Lys Glu Ile Pro Gln Gly Asp Tyr 100105 110 Thr Ile Thr Ala Arg Leu Thr Asn Glu Asp Arg Ala Thr Val Ala Cys115 120 125 Ala Asp Phe Thr Val 130 29 base pairs nucleic acid singlelinear cDNA 4 GACTCCATGG GCGGCGGTGG GAAAGCCTG 29 30 base pairs nucleicacid single linear cDNA 5 GACTAGATCT GGAGCACATG ATAGTAGCAT 30 36 basepairs nucleic acid single linear cDNA 6 GACTGGATCC GCCATCATGA AGGGTTTCACAGCCAC 36 29 base pairs nucleic acid single linear cDNA 7 GACTGGTACCAGCAGCTGCA CTCTTTGGG 29 36 base pairs nucleic acid single linear cDNA 8AGCTAAGCTT CCGCCACCAT GAAGGGTTTC ACAGCC 36 62 base pairs nucleic acidsingle linear cDNA 9 CAGTCTCGAG TTAAGCGTAG TCTGGGACGT CGTATGGGTAGGAGCACATG ATAGTAGCAT 60 TG 62 29 base pairs nucleic acid single linearcDNA 10 GACTGGTACC AGCAGCTGCA CTCTTTGGG 29 514 base pairs nucleic aciddouble linear cDNA 11 NAATTCGCGA GATTTTTCCC AGCTGCAGTG GAGGCGGCGGTGGGAAAGCC TGGCCCACAC 60 ACGTGGTCTG TAGCGACAGG CTTTGGAAGT GCTCTACCAGAGTTGCGATC CATTACAAGA 120 TTTTGGCTTT TCTGTTGAAA AGTGTTCCAA GCAATTAAAATCAAATATCA ACATTAGATT 180 TGGAATTATT CTGAAGGACA TCAAAGAGCT TTTTCTTGACCTAGCTCTCA TGTNTCAAGG 240 CTCATCTGTT TTGAATTTCT CCTATCCCAT CTGTGAGGCGGCTCTGCCAA GTTTTCTTTC 300 TGTGGAAGAA GGAAAGGAGA GCAGATTTAC TATGCTNGGGCTGTCAATAA TNCNGAATTT 360 ACTATTTCCT CANGGGGGAT TACCAGGTTT TGCTGGGACTGTACAATGAA AAACGGTCCA 420 CCGNGGCNGT GCCATGGTAC TATCGNGTGG TCCGACTGTGGCCNTAGGAA AATCACACCA 480 TTGNATTCGG GGNCNCCAGT CCTTGATNAC CNAN 514 457base pairs nucleic acid double linear cDNA 12 CACAGCCACT CTCTTCCTCTGGACTCTAAT TTTNCCCAGC TGCAGTGGAG GCGGCGGTGG 60 GAAAGCCTGG CCCACACACGTGGTCTGTAG CGACANGGCT TGGAAGTGCT CTACCAGAGT 120 TGCGATCCAT TACAAGATTTTGGCTTTTCT GTTGAAAAGT GTTCCAAGCA ATTAAAATCA 180 AATATCAACA TTAGATTTGGAATTATTCTG AGAGAGGACA TCAAAGAGCT TTTTCTTGAC 240 CTAGCTCTCA TGTCTCAAGGCTCATCTGTT TTNAATTTCT CCTATCCCAT CTGTNAGGCG 300 GCTCTGCCCA AGTTTTCTTTCTGTGGAAGA AGGAAAGGAG AGCAGATTTA CTATGCTGGG 360 CCTGTTCAAT AAATCCTGAATTTAACTATT CCTCAGGGAG AATACCAGGT TTTGCTGGAA 420 CTGTACACTG AAAAACGGTCCACCGTGGCC TGTGCCA 457 413 base pairs nucleic acid double linear cDNA 13TTGGTAACNT GTGAGGAAGC ACTCCTAAAA AGTTGAAAAG CAGGTGAGTC TGGTGGGAGG 60CGACCAATTA GAGAAAAAGG ATGGACCCTC AAGACTGCTT AAGGACTGCC AAGAGGCTCC 120TTAGAAACAG ATACATTCGG GGACTTCATT ACATCTGTGG AGTCTGGGGA TGTCTGGTCC 180TGGGACTAAA ATTAGCAGCT GCACTCTTTG GGGGCGCTTC TTTGTAGAGC CTCTCTCTGT 240CATCAGCTGC TTCTCCTCCC ACAGTAGGTT CAGTCAGAGG AGCTTGGAGG TCCCACGAGA 300TGCAGCTGGC TGTGATTTTT GCTACAGGCC ACAGTCAGGA GCACATGATA GTAGCATTGG 360CACAGGCCAC GGTGGACCGT TTTTCAGTGT ACAGTTCCAG CAAAACCTGG GTA 413 320 basepairs nucleic acid double linear cDNA 14 GGACATCAAA GAGCTTTTTCTTGACCTAGC TCTCATGTCT CAAGGCTCAT CTGTTTTGAA 60 TTTCTCCTAT CCCATCTGTGAGGCGGCTCT GCCAAGTTTT CTTTCTGTGG AAGAAGGAAA 120 GGAGAGCAGA TTTACTATGCTGGGCCTGTC AATAATCCTG AATTTACTAT TCCTCAGGGA 180 GAATACCAGG TTTTGCTGGAACTGTACACT GAAAAACGGT CCACCGTGGG CCTGTGNCAA 240 TGCTTACTAT TCATGTGCTCCTGACTGTGG GCCTGTTAGC AAAAANTCAC AGNCAGCTGC 300 ATCTCGTNGG GAACCTTCCA320 264 base pairs nucleic acid double linear cDNA 15 GGCACGAGCCCACCATGAAG GGTTTCACAG CCACTCTCTT CCTCTGGACT CTCATTTTTC 60 CCAGCTGCAGTGGAGGCGGC GGTGGGGAAA GCCTGGCCCA CACACGTGGT CTGTAGCGAC 120 AGNCTTTGGGAAGTGCTCTA CCAGAGTTGC GATCCATTAC AAGATTTTGG CTTTTCTGTT 180 GAAAAGTGTTCCAAGCAATT AAAATCAAAT ATCAACATTA GATTTGGANT TATTCTGAGA 240 GAGGACATCAANGAGCTTTT TTTT 264 249 base pairs nucleic acid double linear cDNA 16GATCGATTAC AAGATNTTGG CTTNTCTGTT GAAAAGTGTT CCAAGCAATT AAAATCAAAT 60ATCAACATTA GATTTGGAAT TATTCTGAGA GAGGACATCA AAGAGCTTTT TCTTGACCTA 120GCTCTCATGT CTCAAGGCTC ATCTGTTTTG ANTTTCTCCT ATCCCATCTG TGAGGCGGCT 180CTGCCNAAGT TTTCTTTCTG TGGNAGAAGG AAANGGGGNC AGNTTTACTT NTTCTTGTNC 240NTTTCNATT 249 60 amino acids amino acid single Not Relevant peptide 17Trp Pro Thr His Val Cys Leu Glu Tyr Ser Cys Asp Pro Gln Asp Phe 1 5 1015 Phe Ser Cys Ser Ile Arg Leu Arg Ile Lys Glu Leu Leu Gly Val Leu 20 2530 Ser Cys Leu Lys Phe Cys Gly Lys Gly Glu Tyr Tyr Gly Pro Ile Pro 35 4045 Gln Gly Tyr Leu Glu Arg Thr Val Ala Cys Ala Thr 50 55 60

What is claimed is:
 1. An isolated nucleic acid molecule comprising apolynucleotide having a nucleotide sequence at least 95% identical to asequence selected from the group consisting of: (a) a nucleotidesequence encoding a polypeptide comprising amino acids from about −20 toabout 142 in SEQ ID NO:2; (b) a nucleotide sequence encoding apolypeptide comprising amino acids from about −19 to about 142 in FIG. 1SEQ ID NO:2; (c) a nucleotide sequence encoding a polypeptide comprisingamino acids from about 1 to about 142 in SEQ ID NO:2; (d) a nucleotidesequence encoding a polypeptide having the amino acid sequence encodedby the cDNA clone contained in ATCC Deposit No. 97825; (e) a nucleotidesequence encoding the mature HOIPS I polypeptide having the amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No. 97825;and (f) a nucleotide sequence complementary to any of the nucleotidesequences in (a), (b), (c), (d) or (e).
 2. An isolated nucleic acidmolecule comprising a polynucleotide which hybridizes under stringenthybridization conditions to a polynucleotide having a nucleotidesequence identical to a nucleotide sequence in (a), (b), (c), (d), (e)or (f) of claim 1 wherein said polynucleotide which hybridizes does nothybridize under stringent hybridization conditions to a polynucleotidehaving a nucleotide sequence consisting of only A residues or of only Tresidues.
 3. An isolated nucleic acid molecule comprising apolynucleotide which encodes the amino acid sequence of anepitope-bearing portion of a HOIPS I polypeptide having an amino acidsequence in (a), (b), (c), (d), or (e) of claim
 1. 4. The isolatednucleic acid molecule of claim 3, which encodes an epitope-bearingportion of a HOIPS I polypeptide selected from the group consisting of:a polypeptide comprising amino acid residues from about 4 to about 9 ofSEQ ID NO:2, a polypeptide comprising amino acid residues from about 13to about 19 of SEQ ID NO:2, a polypeptide comprising amino acid residuesfrom about 23 to about 32 of SEQ ID NO:2, a polypeptide comprising aminoacid residues from about 36 to about 47 of SEQ ID NO:2, a polypeptidecomprising amino acid residues from about 54 to about 63 of SEQ ID NO:2,a polypeptide comprising amino acid residues from about 70 to about 74of SEQ ID NO:2, a polypeptide comprising amino acid residues from about90 to about 100 of SEQ ID NO:2, a polypeptide comprising amino acidresidues from about 105 to about 119 of SEQ ID NO:2, and a polypeptidecomprising amino acid residues from about 125 to about 132 of SEQ IDNO:2.
 5. An isolated nucleic acid molecule comprising a polynucleotidehaving a sequence at least 95% identical to a sequence selected from thegroup consisting of: (a) the nucleotide sequence of a fragment of thesequence shown in SEQ ID NO: 1, wherein said fragment comprises at least50 contiguous nucleotides of SEQ ID NO: 1, provided that said nucleotidesequence is not SEQ ID NO:1, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO:14,SEQ ID NO:15, or SEQ ID NO:16; and (b) a nucleotide sequencecomplementary to a nucleotide sequence in (a).
 6. A method for making arecombinant vector comprising inserting an isolated nucleic acidmolecule of claim 1 into a vector.
 7. A recombinant vector produced bythe method of claim
 6. 8. A method of making a recombinant host cellcomprising introducing the recombinant vector of claim 7 into a hostcell.
 9. A recombinant host cell produced by the method of claim
 8. 10.A recombinant method for producing a HOIPS I polypeptide, comprisingculturing the recombinant host cell of claim 9 under conditions suchthat said polypeptide is expressed and recovering said polypeptide. 11.An isolated HOIPS I polypeptide having an amino acid sequence at least95% identical to a sequence selected from the group consisting of: (a)amino acids from about −20 to about 142 in SEQ ID NO:2; (b) amino acidsfrom about −19 to about 142 in SEQ ID NO:2; (c) amino acids from about 1to about 142 in SEQ ID NO:2; (d) the amino acid sequence of the HOIPS Ipolypeptide having the amino acid sequence encoded by the cDNA clonecontained in ATCC Deposit No. 97825; (e) the amino acid sequence of themature HOIPS I polypeptide having the amino acid sequence encoded by thecDNA clone contained in ATCC Deposit No. 97825; and (f) the amino acidsequence of an epitope-bearing portion of any one of the polypeptides of(a), (b), (c), (d), or (e).
 12. An isolated polypeptide comprising anepitope-bearing portion of the HOIPS I protein, wherein said portion isselected from the group consisting of: a polypeptide comprising aminoacid residues from about −4 to about 9 of SEQ ID NO:2, a polypeptidecomprising amino acid residues from about 13 to about 19 of SEQ ID NO:2,a polypeptide comprising amino acid residues from about 23 to about 32of SEQ ID NO:2, a polypeptide comprising amino acid residues from about36 to about 47 of SEQ ID NO:2, a polypeptide comprising amino acidresidues from about 54 to about 63 of SEQ ID NO:2, a polypeptidecomprising amino acid residues from about 70 to about 74 of SEQ ID NO:2,a polypeptide comprising amino acid residues from about 90 to about 100of SEQ ID NO:2, a polypeptide comprising amino acid residues from about105 to about 119 of SEQ ID NO:2, and a polypeptide comprising amino acidresidues from about 125 to about 132 of SEQ ID NO:2.
 13. The isolatedpolypeptide of claim 11, which is produced or contained in a recombinanthost cell.
 14. The isolated polypeptide of claim 13, wherein saidrecombinant host cell is mammalian.
 15. An isolated nucleic acidmolecule comprising a polynucleotide encoding a HOIPS I polypeptidewherein, except for one to fifty conservative amino acid substitutions,said polypeptide has a sequence selected from the group consisting of:(a) a nucleotide sequence encoding a polypeptide comprising amino acidsfrom about −20 to about 142 in SEQ ID NO:2; (b) a nucleotide sequenceencoding a polypeptide comprising amino acids from about −19 to about142 in FIG. 1 SEQ ID NO:2; (c) a nucleotide sequence encoding a polypeptide comprising amino acids from about 1 to about 142 in SEQ ID NO:2;(d) a nucleotide sequence encoding a polypeptide having the amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No. 97825;(e) a nucleotide sequence encoding the mature HOIPS I polypeptide havingthe amino acid sequence encoded by the cDNA clone contained in ATCCDeposit No. 97825; and (f) a nucleotide sequence complementary to any ofthe nucleotide sequences in (a), (b), (c), (d) or (e).
 16. An isolatedHOIPS I polypeptide wherein, except for one to fifty conservative aminoacid substitutions, said polypeptide has a sequence selected from thegroup consisting of: (a) amino acids from about −20 to about 142 in SEQID NO:2; (b) amino acids from about −19 to about 142 in SEQ ID NO:2; (c)amino acids from about 1 to about 142 in SEQ ID NO:2; (d) the amino acidsequence of the HOIPS I polypeptide having the amino acid sequenceencoded by the cDNA clone contained in ATCC Deposit No. 97825; (e) theamino acid sequence of the mature HOIPS I polypeptide having the aminoacid sequence encoded by the cDNA clone contained in ATCC Deposit No.97825; and (f) the amino acid sequence of an epitope-bearing portion ofany one of the polypeptides of (a), (b), (c), (d), or (e).